Protein and monoclonal antibody specific thereto

ABSTRACT

A novel protein which is useful as a diagnostic means for studies relating to the diagnosis and treatment of cancer (detection of cancer cells, estimation of the malignity, etc.) and for other medicinal and physiological purposes; a gene encoding the same; and an antibody, in particular, a monoclonal antibody specific to the protein. MT-MMP-3, which is a pro MMP-2 activator having the ability to activate pro MMP-2 which is under expression specifically on the surface layer of a human cancer cell and falling within the category of MMP but being different from MT-MMP-1; a DNA containing the base sequence encoding the same; host cells transformed by the DNA; a process for producing a matrix metalloproteinase protein by using the host cells; a monoclonal antibody binding specifically to the matrix metalloproteinase protein; and use of the protein and antibody.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel protein useful as a diagnostictool for studies and researches relating to diagnostic and therapeuticapplications to tumors, including uses in detecting tumor cells,estimating cancer malignancies, etc., and/or useful in other medical andphysiological uses; and to a novel gene encoding said protein. Morespecifically, the present invention relates to a new membrane-typeprotein which is one of the MMPs having the activation capability ofpro-matrix metalloproteinase 2 (pro-MMP-2), i.e. an activator for proMMP-2, provided that said protein is different from the firstmembrane-type matrix metalloproteinase (MT-MMP-1), and to a gene codingfor said protein. The present invention also encompasses a novel matrixmetalloproteinase being specifically expressed in a human tumor cellsurface layer (the instant novel matrix metalloproteinase is named“membrane-type matrix metalloproteinase-3 (MT-MMP-3)”); DNA containing anucleotide sequence coding the protein; a host cell transformed ortransfected with the DNA, a process for producing the matrixmetalloproteinase which comprises using said host cell, a monoclonalantibody capable of specifically binding with the matrixmetalloproteinase protein, and applications of said protein andantibody.

2. Description of the Related Art

An extracellular matrix may block the transfer of tumor cells in theinvasion and metastasis of tumor cells that are present in a primarynest tissue. In order for tumor cells to transfer and invade intotissues, they must deviate from the primary nest and destroy peripheralextracellular matrixes. The metastasis of tumor cells progresses viadestruction of basement membranes, invasion into and effusion from bloodvessels, successful implantation on secondary organs, further growth,etc. The extracellular matrix that blocks tumor metastasis is composedof various complex components, including type IV collagen,proteoglycans, elastin, fibronectin, laminin, heparan sulfate, etc. Afamily of enzymes, generally named “Matrix Metalloproteinase”(hereinafter briefly referred to as “MMP”), with distinct substratespecificities are responsible for the degradation of the extracellularmatrix.

It has been reported that MMP includes fibroblast-type collagenase(MMP-1), 72 kDa gelatinase (referred to as type IV collagenase orgelatinase A; MMP-2), 92 kDa gelatinase (referred to as type IVcollagenase or gelatinase B; MMP-9), stromelysin-1 (MMP-3), matrilysin(MMP-7), neutrophilic collagenase (MMP-8), stromelysin-2 (MMP-10),stromelysin-3 (MMP-11), etc. (Crit. Rev. Oral. Biol. Med., 4: 197 to250, 1993). These MMPs form a family, and the primary structure of geneshas been already reported. The reported amino-acid sequences deducedfrom cDNA data of these MMPs are recognized to be homologous, which areconstituted of an N-terminal signal peptide basically removed duringsecretion and processing, followed by a propeptide domain, a Zn⁺-bindingcatalytic domain, a proline-rich hinge domain composed of 5 to 50 aminoacids, and a C-terminal hemopexin coagulation enzyme-like domain. Thereis no hemopexin-like domain in MMP-7. MMP-2 and MMP-9 include agelatine-binding domain in addition to these.

Among these MMPs, it has been reported many times that type IVcollagenase (MMP-2 and MMP-9) acting on, as a dominant substrate, typeIV collagen that is a principal constituent for basement membranes ishighly expressed in high metastatic tumor cells and there has beensuggested that tumor cells are associated with tumor invasion intobasement membrane invasion (Cell., 64: 327 to 336, 1991). The regulationof MMP activation is believed to be performed in steps including atleast transcription level, a step for converting a proenzyme formwherein its enzymatic activity is latent into an active enzyme form, andcontrols by tissue inhibitor of metalloproteinase (TIMP) being aspecific inhibitor against MMPs, etc. (Trends Genet., 6: 121 to 125,1990).

All of the MMPs are secreted as inactive zymogens. In in vitro studies,activation of MMP-1 and MMP-9 is shown to be produced with serineproteinases such as plasmin, trypsin, cathepsin G. It has also beenreported that activation of MMP-9 is caused by the action of activeMMP-3 (J. Biol. Chem., 267: 3581 to 3584, 1992). However, since MMP-2has no cleavage site sensitive to the above mentioned proteinase,activation of MMP-2 is believed not to be generated thereby (Curr. Opin.Cell Biol., 5: 891 to 897, 1993).

It has also been reported that these MMPs are produced by not only tumorcells but also circumferential fibroblasts and inflammatory cells whichproduce distinct MMPs, respectively (Breast Cancer Res. Treat., 24: 209to 218, 1993; and Curr. Opin. Cell Biol., 5: 891 to 897, 1993).

It has previously been reported that, among them, MMP-2 is expressed infibroblasts at a variety of sites accompanied with remodeling of tissueconstructs and its activation is specifically generated in cancertissues exemplified by lung cancer, in comparison with normal tissue andcancer tissue MMP-2s (Clin., Exp., Metastasis, 11: 183 to 189, 1993). InMMP-9, there is a low frequency that an active type is detected. Inaddition, there is proved in in vitro studies that active MMP-2 islocalized at the apical site of tumor invasion (invadopodia) and it issuggested that the active MMP-2 has an important role on tumor invasion(Cancer Res., 53: 3159 to 3164, 1993; and Breast Cancer Res. Treat., 53:3159 to 3164, 1994).

Under these backgrounds, attention has been focused on the activationmechanism of MMP-2. As described previously, however, activation ofMMP-1 and MMP-9 is mediated by serine proteinases such as trypsin whilethe activation mechanism of MMP-2 is still undisclosed. In particular,an activating factor for MMP-2 remains unidentified. When HT1080 cells(MMP-2 producing cells) are treated with concanavalin A or12-o-tetradecanoylphorbol 13-acetate (TPA), it is known that activeMMP-2 appears in cultured medium, and it is believed that MMP-2activating factors are induced in these cells (J. Natl. Cancer Inst.,85: 1758 to 1764, 1993; and Clin. Exp. Metastasis., 11: 183 to 189,1993). Since this MMP-2 activation is induced by cellular membranefractions and the activation is suppressed by chelating agents or TIMP,the MMP-2 activating factors have been presumed to be a membrane-typeMMP (J. Biol. Chem., 268: 14033 to 14039, 1993).

The present inventors have previously cloned novel MMP genes usinggenetic engineering techniques, and obtained cloned genes coding for anew MMP having a typical transmembrane (TM) domain at the C-terminusthereof and being capable of activating MMP-2 (Nature, 370: 61 to 65,1994). In fact, when this gene is expressed in cultured cells, the geneproducts are localized on the cell membrane without secretion. Thus, thepresent inventors have named such MMP as “membrane-type MMP (MT-MMP)”.

Since, as described above, for MMPs, specifically MMP-2, the active formis found specifically in tumor cells, it is increasingly recognized thatsuch should be targeted by anti-cancer or anti-metastatic drugs. Still,since MMP-2 exists relatively homeostatically as a zymogen in normaltissues, the regulation of MMP-2 activation resides in a process ofactivating it to active enzymes. Therefore, it is considered that theretrieval or identification of activating factors which are keys to thisis extremely important in view of markers in the diagnosis of cancersand in the determination of malignancy and targets of anti-metastaticdrugs against cancers.

In addition, it has been pointed out that MMP-2 may be involved in thecleavage of β-amyloid protein which is associated with the crisis ofAlzheimer's diseases. The β-amyloid protein is a part of amyloid proteinprecursors, ¼ of β-amyloid protein area is included in themembrane-spanning (or transmembrane) area of the amyloid proteinprecursor, and the rest is outside the cells. It has been recentlydisclosed that several metabolic pathways of amyloid protein precursorsexist, one of which is a process including a cleavage of inner sites ofthe β-amyloid protein area with a protease called “α-secretase” and adischarge outside cells. It has been recently found that an amyloidprotein-degrading activity is present in MMP-2, with the possibilitythat MMP-2 would function as α-secretase or an extracellular β-amyloidprotein-degrading enzyme (Nature, 362 : 839, 1993). The β-amyloidprotein is the main component of senile macula observed in the brains ofpatients with Alzheimer's diseases, and forms the core of senile maculaby self-aggregation and deposition thereof. Since functional reductionof β-amyloid protein-degrading enzymes may occur in the brain of thepatient with Alzheimer's diseases, attention is focused on MMP-2. Here,the key is a process for activating MMP-2. The MT-MMP previouslyidentified by the present inventors (newly named “MT-MMP-1” herein) isbelieved to be an activating factor for MMP-2, but the existence ofunknown MMPs such as MT-MMP-1 can be anticipated from the fact that avariety of components exists in the extracellular matrix. The existenceof activating factors for MMP-2, other than MT-MMP-1, is stillundeniable.

SUMMARY OF THE INVENTION

A primary object of the present invention is to provide novel proteinswhich (i) belong to a member of MMPs having the capability of activatingpro MMP-2, (ii) are different from MT-MMP-1, (iii) have the capabilityof activating pro MMP-2, and (iv) are an activator for pro MMP-2; genesencoding the same; processes for producing said novel pro MMP-2activating factor proteins; applications of the protein and gene, etc.

The present inventors have observed that an activating factor(activator) for pro MMP-2 is assumed as a member of membrane-type MMPssince activation of pro MMP-2 is induced by tumor cell membranefractions and the activation is inhibited by chelating agents or TIMP;the present inventors thus have isolated the gene coding for novel MMP-2capable of activating pro MMP-2 in the prior research. However, thepresent inventors have hypothesized the existence of MMP acting as aMMP-2 activator in addition to the above, or MMP biochemically differingfrom the known MMPs. Following various researches using geneticengineering techniques, the present inventors successfully isolated agene coding for MMP that is a novel activating factor for pro MMP-2, andcompleted the present invention.

It has been known that MT-MMP-1 is a member of MMPs capable ofactivating pro MMP-2; however, other activating factors for pro MMP-2have been neither isolated nor identified. The present inventors havenow cloned novel MMP genes, i.e. pro MMP-2 activating factor genes, anddisclosed an entire nucleotide sequence of the gene and an entire aminoacid sequence thereof. The inventors originally named this novel MMP as“MT-MMP-2” (Japanese Patent Application, Nos. Hei 7-200319 (or JP Appln.No. 200319/1995) and 7-200320 (or JP Appln. No. 200320/1995), both filedon Jul. 14, 1995). Later, at the Gordon Research Conference on MatrixMetalloproteinases (Andover, N.H., Jul. 16-21, 1995), it was agreed uponrenaming “MT-MMP-3” (The Journal of Biological Chemistry, Vol. 270, pp.23013-23020 (1995)). Therefore, the instant “MT-MMP-3” indicates thesubstance identical with MT-MMP-2 as described in Japanese PatentApplication Nos. 7-200319 and 7-200320.

The present invention relates to novel proteins, i.e. MT-MMP-3 andanalogs thereof. Further, the present invention relates to novel DNAsequences coding for all or part of MT-MMP-3, to vectors having such DNAsequences, and to host cells transformed or transfected with suchvectors. The present invention also includes the production ofrecombinant MT-MMP-3 and uses of said recombinant MT-MMP-3. The presentinvention relates to antibodies which specifically bind with MT-MMP-3.In another aspect, the present invention relates to reagents formeasurement or assay which contain said product and to detecting,measuring or assaying methods using such reagents. In particular,methods for detecting or measuring MT-MMP-3 in vivo and in vitro areprovided.

The present invention relates to (1) proteins or a salt thereof which(i) belong to a member of MMPs capable of activating pro MMP-2 but arenot MT-MMP-1, (ii) are an activator for pro MMP-2 and (iii) have anactivity identical with or substantially equivalent tonaturally-occurring MT-MMP, or a salt thereof; (2) characteristicpartial peptides of said protein or a salt thereof; (3) genes (forexample, nucleic acids including DNA, RNA, etc.) coding for said proteinor peptide; (4) vectors or plasmids which contain said gene operablywith gene recombination techniques; (5) host cells transformed ortransfected with such vectors or the like; (6) processes for producingsaid protein or a salt thereof which comprises culturing saidtransformed or transfected host cell (transformant or transfectant); (7)antibodies (in particular, monoclonal antibodies) obtained using amember selected from the group consisting of the protein or a saltthereof thus obtained in the above process and the characteristicpartial peptide of the protein or a salt thereof thus obtained in theabove process; (8) hybridoma cells which produce the antibody; and (9)measuring (assaying) and/or diagnostic means (i) using the isolated gene(including, for example, DNA, RNA, etc.) as a probe or (ii) using theantibody.

Particularly, the present invention relates to (1) proteins which (i)belong to a member of MMPs capable of activating pro MMP-2, (ii) are anactivator for pro MMP-2 but different from MT-MMP-1 and (iii) have anactivity identical with or substantially equivalent to native MT-MMP-3,or a salt thereof; (2) characteristic partial peptides of said proteinor a salt thereof; (3) genes (including, for example, DNA, RNA, etc.)coding for said protein or peptide; (4) vectors or plasmids wherein saidgene is contained operably with gene recombination techniques; (5) hostcells transformed or transfected with such a vector or the like; (6)processes for producing said protein or a salt thereof which comprisesculturing said transformed or transfected host (transformant ortransfectant)); (7) antibodies (in particular, monoclonal antibodies)obtained using a member selected from the group consisting of saidprotein or a salt thereof thus obtained and the unique partial peptideof said protein or a salt thereof thus obtained; (8) hybridoma cellswhich produce the antibody; and (9) measurement (assay) and/or diagnosismeans (i) using the isolated gene (including, for example, DNA, RNA,etc.) as a probe or (ii) using the antibody.

Preferably, the present invention is related to MT-MMP-3 or a saltthereof which has (i) an amino acid sequence represented by SEQ ID NO: 2in the Sequence Listing or (ii) an amino acid sequence substantiallyequivalent to SEQ ID NO: 2.

The present invention provides:

(1) a protein or a salt thereof, which (i) belongs to a member of MMPshaving the activation capability of pro MMP-2, (ii) has an activityidentical with or substantially equivalent to naturally-occurringMT-MMP, and (iii) is a pro MMP-2 activating factor, excluding MT-MMP-1;

(2) the protein according to above (1), wherein the protein has abiological property or primary structural conformation identical with orsubstantially equivalent to that of native MT-MMP-3 or a salt thereof;

(3) the protein according to above (1) or (2), wherein a C-terminal areaof the protein has (i) an amino acid sequence from Ala⁵⁶⁴ to Phe⁵⁸⁷ inthe sequence represented by SEQ ID NO: 2 in the Sequence Listing or (ii)an amino acid sequence substantially equivalent thereto;

(4) the protein according to any of above (1) to (3), wherein theprotein is MT-MMP-3 or a salt thereof which has (i) an amino acidsequence represented by SEQ ID NO: 2 in the Sequence Listing or (ii) anamino acid sequence equivalent thereto;

(5) the protein according to any of above (1) to (4), wherein theprotein is the product of prokaryotic or eukaryotic expression of anexogenous DNA sequence;

(6) the protein according to any of above (1) to (5), wherein theprotein has (i) the amino acid sequence of SEQ ID NO: 2 in the SequenceListing or (ii) the substantially same amino acid sequence;

(7) a partial peptide (or a peptide fragment) of the protein accordingto any of above (1) to (6) or a salt thereof;

(8) a nucleic acid comprising a nucleotide sequence coding for theprotein or the partial peptide according to any of above (1) to (7);

(9) the nucleic acid according to above (8) which is a DNA gene having anucleotide sequence coding for MT-MMP-3 according to any of above (2) to(4);

(10) the nucleic acid according to above (8) or (9), having (i) an openreading frame region of the nucleotide sequence represented by SEQ IDNO: 1 in the Sequence Listing or (ii) a nucleotide sequence having anactivity substantially equivalent thereto;

(11) a vector comprising the nucleic acid according to any of above (8)to (10);

(12) a transformant or transfectant harboring (i) the nucleic acidaccording to any of above (8) to (10) or (ii) the vector according toabove (11);

(13) a process for producing the protein according to any of above (1)to (6) or a partial peptide thereof, which comprises:

(i) culturing the transformant or transfectant according to above (12)in a nutrient medium capable of growing said transformant ortransfectant, and

(ii) producing, as a recombinant species, the protein according to anyof above (1) to (6) or a partial peptide thereof, including MT-MMP-3 ora salt thereof;

(14) an antibody against (a) a protein or a salt thereof which (i)belongs to a member of MMPs having the activation capability of proMMP-2, (ii) has an activity identical with or substantially equivalentto naturally- occurring MT-MMP, and (iii) is a pro MMP-2 activatingfactor, excluding MT-MMP-1, or (b) a partial peptide of said protein ora salt thereof;

(15) the antibody according to above (14), wherein the antibody isagainst the protein which has an activity or a primary structuralconformation identical with or substantially equivalent to that ofMT-MMP-3 or a salt thereof;

(16) the antibody according to above (14) or (15), wherein the antibodyis against the protein that is MT-MMP-3 or a salt thereof having (i) anamino acid sequence represented by SEQ ID NO: 2 in the Sequence Listingor (ii) an amino acid sequence substantially equivalent thereto;

(17) the antibody according to any of above (14) to (16), wherein theantibody is against the protein which is a product obtained byexpressing a foreign DNA sequence in prokaryotic or eukaryotic cells;

(18) the antibody according to any of above (14) to (17), wherein theantibody is against the protein which has (i) the amino acid sequence ofSEQ ID NO: 2 in the Sequence Listing or (ii) the substantially sameamino acid sequence;

(19) the antibody according to any of above (14) to (18), wherein theantibody is against a partial peptide of the protein or a salt thereof;

(20) the antibody according to any of above (14) to (19), wherein theantibody is an anti-serum;

(21) the antibody according to any of above (14) to (19), wherein theantibody is monoclonal;

(22) the antibody according to any of above (14) to (19) and (21), whichis a monoclonal antibody against MT-MMP-3 or a salt thereof;

(23) a method for producing an antibody against (a) a protein or a saltthereof which (i) belongs to a member of MMPs having the activationcapability of pro MMP-2, (ii) has an activity identical with orsubstantially equivalent to naturally-occurring MT-MMP, and (iii) is apro MMP-2 activating factor, excluding MT-MMP-1, or (b) a partialpeptide of said protein or a salt thereof, which comprises employing anantigen selected from the group consisting of said protein, said partialpeptide and a salt thereof to raise the antibody thereagainst;

(24) a method for producing the antibody according to above (21) or(22), which comprises

(A) fusing an antibody-producing cell obtained from an immunized animalwith an immortal cell, wherein said antibody is against (a) a protein ora salt thereof which (i) belongs to a member of MMPs having theactivation capability of pro MMP-2, (ii) has an activity identical withor substantially equivalent to naturally-occurring MT-MMP, and (iii) isa pro MMP-2 activating factor, excluding MT-MMP-1, or (b) a partialpeptide of said protein or a salt thereof and said animal is immunizedwith the protein, the partial peptide or a salt thereof, and

(B) selecting an immortal hybrid cell capable of an antibody against aprotein including MT-MMP-3;

(25) a method for detecting and/or measuring MT-MMP-3, which comprisesusing (A) a reagent selected from the group consisting of (a) a proteinor a salt thereof which (i) belongs to a member of MMPs having theactivation capability of pro MMP-2, (ii) has an activity identical withor substantially equivalent to naturally-occurring MT-MMP, and (iii) isa pro MMP-2 activating factor, excluding MT-MMP-1, and (b) a partialpeptide of said protein or a salt thereof, or (B) a reagent selectedfrom the group consisting of the antibodies according to any of above(14) to (22);

(26) a labeled antibody against MT-MMP-3 for the method for detectingand/or measuring MT-MMP-3 (the detection and/or measurement of MT-MMP-3)according to above (25);

(27) a labeled protein or a salt thereof, for the method for detectingand/or measuring MT-MMP-3 (the detection and/or measurement of MT-MMP-3)according to above (25), wherein said labeled protein (i) belongs to amember of MMPs having the activation capability of pro MMP-2, (ii) hasan activity identical with or substantially equivalent tonaturally-occurring MT-MMP, and (iii) is a pro MMP-2 activating factor,excluding MT-MMP-1, or a labeled partial peptide of said protein or asalt thereof, for the method according to above (25);

(28) a labeled nucleic acid for detection and/or measurement of MT-MMP-3expressing cells and/or tissues, wherein said nucleic acid encodes (A) aprotein which (i) belongs to a member of MMPs having the activationcapability of pro MMP-2, (ii) has an activity identical with orsubstantially equivalent to naturally-occurring MT-MMP, and (iii) is apro MMP-2 activating factor, excluding MT-MMP-1, or (B) a partialpeptide of said protein; and

(29) the nucleic acid according to above (28), which is a probe forhybridization.

In particular, the present invention provides:

(30) MT-MMP-3 or a salt thereof which has an amino acid sequencerepresented by SEQ ID NO: 2 in the Sequence Listing or an amino acidsequence substantially equivalent thereto;

(31) a partial peptide of MT-MMP-3 or a salt thereof according to above(30);

(32) a DNA gene comprising a nucleotide sequence coding for MT-MMP-3according to above (30);

(33) the DNA gene according to above (32), which has a nucleotidesequence represented by SEQ ID NO: 1 in the Sequence Listing;

(34) a vector comprising the gene according to above (32);

(35) a transformant (or transformed cell) harboring (i) the geneaccording to above (32) or (ii) the vector according to above (34);

(36) a process for producing MT-MMP-3 or a salt thereof, which comprisesculturing the transformant according to above (35) in a nutrient mediumcapable of growing said transformant to produce, as a recombinantprotein, said MT-MMP-3 or a salt thereof;

(37) a process for producing an antibody against MT-MMP-3 or a saltthereof, which comprises using an antigen selected from the groupconsisting of MT-MMP-3 or a salt thereof according to above (30) and apartial peptide of said MT-MMP-3 or a salt thereof to raise the antibodythereagainst;

(38) an antibody against MT-MMP-3 according to above (31);

(39) the antibody (anti-MT-MMP-3 antibody) according to above (38),which is anti-serum;

(40) the antibody (anti-MT-MMP-3 antibody) according to above (38),which is monoclonal;

(41) a process for producing a monoclonal antibody against MT-MMP-3(monoclonal anti-MT-MMP-3 antibody; anti-MT-MMP-3 mAb) according toabove (40), which comprises fusing an anti-MT-MMP-3 antibody-producingcell with an immortal cell and selecting an immortal hybrid cell(hybridoma cell) capable of producing anti-MT-MMP-3 mAb, wherein saidanti-MT-MMP-3 antibody-producing cell is obtained from an animalimmunized with a member selected from the group consisting of MT-MMP-3or a salt thereof according to above (30) and a partial peptide of saidMT-MMP-3 or a salt thereof;

(42) a method for detecting and/or measuring MT-MMP-3, which comprisesusing (A) a reagent selected from the group consisting of MT-MMP-3 or asalt thereof according to above (30) and a partial peptide of saidMT-MMP-3 or a salt thereof, or (B) a reagent selected from the groupconsisting of anti-MT-MMP-3 antibodies according to above (38);

(43) Labeled MT-MMP-3 or a salt thereof, or a labeled partial peptide ofMT-MMP-3, for the method for detecting and/or measuring MT-MMP-3according to above (42); and

(44) a labeled antibody against MT-MMP-3 (labeled anti-MT-MMP-3antibody) for the method for detecting and/or measuring MT-MMP-3according to above (42).

DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1E illustrate the domain structure of MT-MMP-3 according tothe present invention, in comparison with the known MMP family members:MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MT-MMP-1.An alignment of amino acid sequences of the MMP family members is shownwherein the homology among the amino acid sequence of MT-MMP-3 and thereported amino acid sequences of the known MMP family members: MMP-1,MMP-2, MMP-3, MMP-7, MMP-8, MMP-10, MMP-11, and MT-MMP-1 is compared.Each amino acid residue is indicated by a conventional single charactersymbol, and numbered, provided that the N-terminus of pre-type proteinsis designated as the first amino acid residue.

FIG. 2 is photographs showing the electrophoretic results of Northernblotting.

A: RNA blot analysis of MT-MMP-3 mRNA in various human tissues byNorthern blotting.

B: RNA blot analysis of MT-MMP-3 mRNA in various cultured humanmalignant cell lines by Northern blotting.

FIG. 3 is a photograph showing the electrophoretic results ofimmunoprecipitation of cell lysates and conditioned culture mediumwherein MT-MMP-3 cDNA was expressed in COS-1 cells and MT-MMP-3 geneproducts (MT-MMP-3 proteins) were examined.

In autoradiography, MT-MMP-3 protein (64 kDa) and TIMP-1 protein (28kDa) are indicated by arrows: ▴ and Δ respectively.

FIG. 4 is a photograph showing the electrophoretic results of the studythat the fusion protein having a continuous sequence withTIMP-1/hydrophobic amino acid stretch at the C-terminus of MT-MMP-3 wasprepared to examine the role of the continuous sequence composed of thehydrophobic amino acids at the C-terminus of MT-MMP-3 as a transmembrane(TM) domain.

The fusion proteins (chimeric proteins) constructed by gene engineeringtechniques were expressed in COS-1 cells and cell lysates andconditioned culture medium thereof were examined. The electrophoreticresults detected by autoradiography are shown.

FIG. 5 is photographs showing the results of immunofluorescence stainingwherein the fusion protein having a continuous sequence withTIMP-1/hydrophobic amino acid stretch at the C-terminus of MT-MMP-3 wasprepared to examine whether the continuous sequence composed of thehydrophobic amino acids at the C-terminus of MT-MMP-3 functions as atransmembrane (TM) domain.

Biological figures observed by immunofluorescence staining when thechimeric proteins having the continuous sequence with TIMP-1/hydrophobicamino acid stretch were expressed in COS-1 cells.

FIGS. 6A and B is photographs showing the electrophoretic results ofzymography analysis of activation of pro MMP-2 by MT-MMP-3 expression.

A: Activation of pro MMP-2 in COS-1 cells, wherein COS-1 cellscotransfected with MT-MMP-3 cDNA and pro MMP-2 cDNA.

B: Activation of pro MMP-2 by MT-MMP-3 and effect of TIMP-1 and TIMP-2in HT 1080 cells into which MT-MMP-3 cDNA was cotransfected.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides (1) a protein or a salt thereof (i) thatis a member of MMPs capable of activating pro MMP-2 but is not MT-MMP-1,(ii) that has an activity identical with or substantially equivalent tonative MT-MMP (for example, MT-MMP-3) which is a pro MMP-2 activationfactor; (2) a specific partial peptide of that protein or a saltthereof; (3) a gene (such as DNA or RNA) coding for the same; (4) avector or plasmid containing the gene operably by gene recombinationtechniques; (5) a host cell transformed by such a vector; and (6) amethod for producing the protein or a salt thereof by culturing the hostcell; (7) an antibody (in particular a monoclonal antibody) obtainedusing a species selected from the group consisting of the protein thusobtained or a salt thereof and partial peptides (peptide fragments)unique thereto or a salt thereof; (8) a hybridoma cell producing theantibody, and (9) measurement and diagnosis means using as a probe theisolated gene, such as DNA or RNA, or using the antibody.

More particularly, the present invention provides (i) MT-MMP-3 or a saltthereof which has the amino acid sequence of SEQ ID NO: 2 in theSequence Listing. The MT-MMP-3 of the present invention may includethose that are pro MMP-2 activating factors and have a new amino acidsequence as long as they are members of MMPs capable of activating proMMP-2 but different from MT-MMP-1 and are capable of activating proMMP-2. More preferably, the MT-MMP-3 of the present invention includesall substances having an amino acid sequence identical with orsubstantially equivalent to the amino acid sequence of SEQ ID NO: 2 inthe Sequence Listing. Furthermore, the MT-MMP-3 of the present inventionmay have (i) as a pre portion, part or all of the amino acid sequenceranging from the first amino acid residue: Met to the 21st amino acidresidue: Phe, and/or (ii) as a pro portion part or all of the amino acidsequence ranging from the 22nd amino acid residue: Phe to the 119thamino acid residue: Arg. All of MT-MMP-3 that have such a sequence maybe included herein.

The MT-MMP-3 can be encoded by a nucleotide sequence comprising a regionranging between ATG from the 113th to 115th nucleotide residues of SEQID NO: 1 in the Sequence Listing and GTG from the 1931st to 1933rdnucleotide residues (termination codon: TGA from the 1934th to 1936thnucleotide residues may be replaced with TAA or TAG), and can also beencoded by any DNA sequence containing a nucleotide sequence homologousto the above nucleotide sequence but different from the MT-MMP-1sequence as long as it is equivalent to a sequence for a species capableof activating pro MMP-2. The MT-MMP-3 nucleotide sequences can bemodified (by addition, deletion, substitution), and those thus modifiedmay be included herein.

The DNA containing a nucleotide sequence represented by SEQ ID NO: 1 oran equivalent thereof according to the present invention may be clonedand obtained, for example, by the following techniques:

It should be noted that gene recombination techniques may be conducted,for example, by the methods disclosed in T. Maniatis et al., “MolecularCloning”, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.T. (1989); Nippon Seikagaku Kai (Biochemical Society of Japan) ed.,“Zoku-Seikagaku Jikken Kouza 1, Idenshi Kenkyuho II (Lectures onBiochemical Experiments (Second Series; 1), Methods for Gene Study II)”,Tokyo Kagaku Dojin, Japan (1986); Nippon Seikagaku Kai (BiochemicalSociety of Japan) ed., “Shin-Seikagaku Jikken Kouza 2, Kakusan III(Kumikae DNA Gijutsu) (New Lectures on Biochemical Experiments 2,Nucleic Acids III (Recombinant DNA Technique))”, Tokyo Kagaku Dojin,Japan (1992); R. Wu (ed.), “Methods in Enzymology”, Vol. 68, AcademicPress, New York (1980); R. Wu et al. (ed.), “Methods in Enzymology”,Vols. 100 & 101, Academic Press, New York (1983); R. Wu et al. (ed.),“Methods in Enzymology”, Vols. 153, 154 & 155, Academic Press, New York(1987), etc. as well as by techniques disclosed in the references citedtherein, the disclosures of which are hereby incorporated by reference,or by the substantially same techniques as they disclose or modifiedtechniques thereof. Such techniques and means may also be those whichare individually modified/improved from conventional techniquesdepending upon the object of the present invention.

mRNA samples can be isolated from various human tissues (placenta, oraltumor, lung cancer, etc.), culture cells (human fibrosarcoma HT1080 cellline, human monocytic leukemia U937 cell line, etc.) and the like. Inparticular, mRNA can preferably be isolated from a human oral tumor cell(oral malignant melanoma). Although, in an embodiment, mRNA may beisolated with a method known in the art or by the substantially samemethod as it is or modifications thereof, the isolation and purificationof mRNA can be conducted by methods disclosed in, for example, T.Maniatis, et al., “Molecular Cloning”, 2nd Ed., Chapter 7, Cold SpringHarbor Laboratory, Cold Spring Harbor, N. T. (1989); L. Grossman, et al.ed., “Methods in Enzymology”, Vol. 12, Parts A & B, Academic Press, NewYork (1968); S. L. Berger et al. ed., “Methods in Enzymology”, Vol. 152,p. 33 & p. 215, Academic Press, New York (1987); Biochemistry, 18,5294-5299, 1979; etc., the disclosures of which are hereby incorporatedby reference. Examples of such mRNA isolating and purifying techniquesare a guanidine-cesium chloride method, a guanidine thiocyanate method,a phenol method, etc. If necessary, the resulting total RNA may besubjected to a purification process using an oligo(dT)-cellulose column,etc. to give poly(A)⁺mRNA.

cDNAs are prepared by using, as a template, the resulting mRNA and areverse transcriptase, etc. The reverse transcriptase synthesis of cDNAusing mRNA may be carried out by standard techniques known in the art,by the substantially same techniques or by modified techniques thereof.Detailed techniques are found in, for example, H. Land et al., “NucleicAcids Res.”, Vol. 9, 2251 (1981); U. Gubler et al., “Gene”, Vol. 25,263-269 (1983); S. L. Berger et al. ed., “Methods in Enzymology”, Vol.152, p. 307, Academic Press, New York (1987); etc., the disclosures ofwhich are hereby incorporated by reference.

Then, based upon the cDNA thus prepared, cDNA libraries can beconstructed. Besides the technique using a phage vector, transformationsof host cells including Escherichia coli may be conducted according totechniques known in the art, such as a calcium technique and arubidium/calcium technique, or the substantially same methods (D.Hanahan, J. Mol. Biol., Vol. 166, p. 557 (1983), etc.). Variouscommercially available cDNA libraries derived from human tissues (forexample, obtainable by CLONTECH, etc.) can also be used directly. Apolymerase chain reaction (PCR) is conducted using the prepared cDNA asa template. In an embodiment, primers are synthesized which havedegenerate oligonucleotides designed from highly conserved regionsselected from amino acid sequences in a family of known MMPs.Preparation of primers may be carried out by techniques which are knownin the art. For example, the primers may be synthesized by means of aphosphodiester method, a phosphotriester method, a phosphoamiditemethod, etc. using an automatic DNA synthesizer. The PCR amplificationis carried out using said primers and the template cDNA thus prepared.The PCR may be carried out by techniques known in the art or by methodssubstantially equivalent thereto or modified techniques. The reactionmay be conducted by the methods disclosed, for example, in R. Saiki, etal., Science, Vol. 230, pp. 1350 (1985); R. Saiki, et al., Science, Vol.239, pp. 487 (1985); and PCR Technology, Stockton Press; etc., thedisclosures of which are hereby incorporated by reference.

The resulting PCR products are cloned, and sequenced. As a result, DNAfragments having a novel MMP gene sequence are acquired. Sequencing ofnucleotide sequences may be carried out by a dideoxy technique (such asan M13 dideoxy method), a Maxam-Gilbert method, etc. or may be carriedout using a commercially available sequencing kit such as a Taqdyeprimer cycle sequencing kit or an automated nucleotide sequencer suchas a fluorescent DNA sequencer. In particular, cDNA librariesconstructed from various human tissues (placenta, oral tumors, lungcancers, etc.) or culture cells (human fibrosarcoma HT1080 cell line,human monocytic leukemia U937 cell line, etc.) are screened using theDNA fragment as a probe, and the target DNA can be isolated bysequencing of nucleotide sequences. Preferably, a human placenta cDNAlibrary is screened, a detected DNA is sequenced, and the target DNA isisolated and identified. Labeling of probes, etc. with a radioisotope,etc., may be carried out using a commercially available labeling kitsuch as a random primed DNA labeling kit (Boehringer Mannheim).

The detailed description thereof is given below.

The inventors designed and synthesized the following:

5′ primer having the following sequence:

5P-4 (SEQ ID NO: 3)

SGNVVNGCWGAYATMRTSAT

wherein S═C or G, N═A, C, G or T, V═A, C, or G, W═A or T, Y═C or T, M═Aor C, and R═A or G; mixed bases; and 3′ primer having the followingsequence:

3P-2 (SEQ ID NO: 4)

YTCRTSNTCRTCRAARTGRRHRTCYCC

wherein Y═C or T, R═A or G, S═C or G, N═A, C, G or T, and H═A, C or T;mixed bases,

based on highly conserved amino acid sequences GEADILV (SEQ ID NO: 10)and GDAHFDDDE (SEQ ID NO: 10), selected from the catalytic enzyme domainamong the known MMP family.

In the above-mentioned sequences, symbols (S, N, V, W, Y, M, R, and H)indicate the incorporation of plural bases, leading to multipleoligonucleotides in the primer preparation. In other words, SEQ ID NO: 3and SEQ ID NO: 4 are degenerate nucleotide primers.

Primers can be designed, synthesized, and used based on amino acidsequences in the area specific to the MMP family.

PCR was carried out using these primers and the cDNA library preparedfrom a human oral malignant melanoma. The obtained PCR products having asize (90 to 120 b.p.) expected from the primer design were sub-clonedand sequenced. As a result, there were obtained DNA fragments with anovel sequence homologous to the known MMPs, other than PCR productshaving a sequence identical with either MMP-1 or MMP-9.

Similarly, by using these primers and cDNA libraries derived fromvarious human cells, PCR products with a novel sequence homologous tothe known MMPs may be searched, other than PCR products with the samesequence as that of either MMP-1 or MMP-9.

The 93 b.p. DNA fragment was employed as a probe to screen for a humanplacenta cDNA library. As a result, 2.1-kilobase pair DNA fragments wereobtained. The obtained DNA fragments were sequenced and the nucleotidesequence of SEQ ID NO: 1 was determined.

The same nucleotide sequence as that represented by SEQ ID NO: 1 doesnot exist in GENE BANK/EMBL DNA Data Base. Therefore, it has beenrecognized that DNA having the nucleotide sequence of SEQ ID NO: 1 isabsolutely novel.

The nucleotide sequence of the above mentioned clone possessing anucleotide sequence represented by SEQ ID NO: 1 has a 3′non-translational sequence together with an open reading framepotentially coding for a 607 amino acid protein. It has been recognizedthat a deduced signal sequence follows immediately downstream of theinitiation codon and a hydrophobic domain which is composed of aligned24 amino acid residues with higher hydrophobicity and is characteristicof membrane-type proteins is present at the C-terminal area from the564th to 587th amino acid residues.

The novel MMP thus obtained has been named “MT-MMP-3” (the inventorsfirst called it as “MT-MMP-2” in Japanese Patent Application Nos.7-200319 and 7-200320, both filed on Jul. 14, 1995; however, theinventors has agreed to rename it as “MT-MMP-3”, based on the agreementsin the conference of Gordon Research Conference on MatrixMetalloproteinases (Andover, N.H. Jul. 16-21, 1995)).

MT-MMP-3 gene products are confirmed using suitable animal cells, suchas COS-1 cells, transfected with the MT-MMP-3 gene. The foreign gene canbe introduced into mammal animal cells with known methods in the art orwith methods substantially similar thereto, including a calciumphosphate technique (for example, F. L. Graham et al., “Virology”, Vol.52, pp. 456 (1973), etc.), a DEAE-dextran technique (for example, D.Warden et al., “J. Gen. Virol.”, Vol. 3, pp. 371 (1968), etc.), anelectroporation technique (for example, E. Neumann et al., “EMBO J”,Vol. 1, pp. 841 (1982), etc.), a microinjection technique, a liposometechnique, a virus infection technique, a phage particle technique, etc.

Thus, the gene products which were produced by animal cells transfectedwith the MT-MMP-3 gene were examined by means of immunoprecipitationexperiments using monoclonal anti-MT-MMP-3 antibodies. As a result, a 64kDa protein was immunologically precipitated from the lysate of cellstransfected with the MT-MMP-3 gene while no corresponding protein wasdetected on the culture medium. In other words, it is suggested thatMT-MMP-3 gene products are expressed in the cell surface layer withoutbeing secreted.

The MT-MMP-3 protein has been examined for the homology with thereported amino acid sequences of the known MMP family. As shown in FIGS.1A to 1E, it is revealed that MT-MMP-3 has high homology to the knownMMP family. The MT-MMP-3 protein maintains the sequence at or near theprocessing site for conversion of a precursor form to a mature form(corresponding to the sequence conserved in the MMP family) as well asthe sequence of the active site best. In addition, the propeptide domaincharacteristic of the primary structure of MMPs, the Zn⁺ bindingcatalytic domain, the proline-rich hinge domain, and the C-terminalhemopexin coagulation enzyme-like domain are also well conserved in theMT-MMP-3 protein.

Similarly to MT-MMP-1 (the inventors rename the previously isolated andidentified MT-MMP as “MT-MMP-1” in order to distinguish MT-MMP-3therefrom), MT-MMP-3 has a sequence composed of aligned hydrophobicamino acids in the C-terminal region. It is therefore suggested thatMT-MMP-3 is a membrane-type MMP. Such a sequence with alignedhydrophobic amino acids does not exist in the other MMP family members.In fact, when fusion proteins in which the aligned sequence composed ofthe hydrophobic amino acid residues is fused with a secretory protein bygenetic engineering are constructed and expressed in culture cells,secretion of the fusion proteins was suppressed and expressed on thecell membranes. As a result, the aligned sequence composed of thehydrophobic amino acids is shown to function as a transmembrane (TM)domain.

Therefore, it is apparent that MT-MMP-3 gene codes for a novel MMPprotein. Consequently, recombinant plasmids produced using MT-MMP-3 geneare all novel recombinant products, and transformants transformed ortransfected with the plasmid are novel.

Any plasmid into which the MT-MMP-3 gene is incorporated may be used aslong as said DNA can be expressed in host cells conventionally used ingene engineering techniques (such as procaryotic host cells includingEscherichia coli, Bacillus subtilis, etc. and eucaryotic host cellsincluding yeasts, CHO cell, and insect host cells such as Sf2). In sucha sequence of the plasmid, it is possible, for example, to incorporatecodons suitable for expressing the cloned DNA in selected host cells orto construct restriction enzyme sites. It is also possible to havecontrol sequences, promotion sequences, etc. for facilitating theexpression of the aimed gene; linkers, adaptors, etc. useful forligating the aimed gene; sequences useful in controlling resistance toantibiotics or in controlling metabolism or in selection; and the like.

Preferably, suitable promoters may be used. For example, such promotersmay include tryptophan (trp) promoter, lactose (lac) promoter,tryptophan-lactose (tac) promoter, lipoprotein (lpp) promoter, λ phageP_(L) promoter, etc. in the case of plasmids where Escherichia coli isused as a host; SV40 late promoter, MMTV LTR promoter, RSV LTR promoter,CMV promoter, SRα promoter, etc. in the case of plasmids where an animalcell is used as a host; and GAL1, GAL10 promoters, etc. in the case ofplasmids where yeast is used as a host.

Examples of the plasmid suitable for host Escherichia coli are pBR322,pUC18, pUC19, pUC118, pUC119, pSP64, pSP65, pTZ-18R/-18U, pTZ-19R/-19U,pGEM-3, pGEM-4, pGEM-3Z, pGEM-4Z, pGEM-5Zf(−), pbluescript KS™(Stratagene), etc. Examples of the plasmid vector suitable forexpression in Escherichia coli are pAS, pKK223 (Pharmacia), pMC1403,pMC931, pKC30, etc. The plasmid for host animal cells may include SV40vector, polyomavirus vector, vaccinia virus vector, retrovirus vector orthe like. Examples of the plasmid for host animal cells are pcD, pcD-SRα, CDM8, pCEV4, pME18S, pBC12BI, pSG5 (Stratagene) or the like. Examplesof the plasmid for host yeasts are YIp vector, YEp vector, YRp vector,YCp vector, etc., including pGPD-2, etc. Escherichia coli host cells mayinclude those derived from Escherichia coli K12 strains, such as NM533XL1-Blue, C600, DH1, HB101 and JM109.

In the case where the host cells are animal cells, they may include COS7cells, COS-1 cells, and CV-1 cells derived from African green monkeyfibroblasts, COP cells, MOP cells, and WOP cells derived from mousefibroblasts, CHO cells and CHO DHFR⁻ cells derived from chinese hamster,human HeLa cells, C127 cells derived from mouse cells, NIH 3T3 cellsderived from mouse cells, etc. The insect cells may include bombyx morilarva, bombyx mori culture cells such as BM-N cells, etc. wherein bombyxmori nuclear polyhedrosis virus is employed as a vector.

In the gene engineering techniques of the present invention, it ispossible to use various restriction enzymes, reverse transcriptases, DNAmodifying and degrading enzymes which are used for modifying orconverting a DNA fragment to a structure suitable for cloning, DNApolymerases, terminal nucleotidyltransferases, DNA ligases; etc., whichare known or common in the art. Examples of the restriction enzyme arethose disclosed in R. J. Roberts, “Nucleic Acids Res.”, Vol. 13, r165(1985); S. Linn et al. ed., “Nucleases”, p. 109, Cold Spring HarborLab., Cold Spring Harbor, New York, 1982; etc., the disclosures of whichare hereby incorporated by reference. Examples of the reversetranscriptase are those derived from mouse Moloney leukemia virus(MMLV), from avian myeloblastosis virus (AMV), etc. Particularly, RNaseH-deficient reverse transferase or the like is preferably used. Examplesof the DNA polymerase are Escherichia coli DNA polymerase, Klenowfragment which is a derivative of E. coli DNA polymerase, E. coli phageT4 DNA polymerase, E. coli phage T7 DNA polymerase, thermoduric bacteriaDNA polymerase, etc.

The terminal nucleotidyltransferase includes TdTase capable of adding adideoxynucleotide (dNMP) to a 3′-OH terminal, as disclosed in R. Wu etal. ed., “Methods in Enzymology”, Vol. 100, p. 96, Academic Press, NewYork (1983). The enzyme for modifying and decomposing DNA includesexonuclease, endonuclease, etc. Examples of such enzymes are snake venomphosphodiesterase, spleen phosphodiesterase, E. coli DNA exonuclease I,E. coli DNA exonuclease III, E. coli DNA exonuclease VII, λ exonuclease,DNase I, nuclease S1, Micrococcus nuclease, etc. Examples of the DNAligase are E. coli DNA ligase, T4 DNA ligase, etc.

The vector (or vehicle) which is suitable for cloning DNA genes andconstructing DNA libraries includes plasmid, λ phage, cosmid, P1 phage,F factor, YAC, etc. Preferred examples of such vectors are vectorsderived from λ phage, such as Charon 4A, Charon 21A, λ gt10, λ gt11, λDASHII, λ FIXII, λ EMBL3 and λ ZAPII™ (Stratagene), etc.

Further, by relying on the nucleotide sequence of MT-MMP-3 geneaccording to the present invention, equivalent proteins or derivatives(or analogs) thereof wherein the amino acid sequence of MT-MMP-3 isaltered may be produced with conventional gene technological methods.Such alterations includes substitution, deletion, insertion, transfer oraddition of one or more amino acid residues, etc. Such methods formutation, conversion, and/or modification may also include thosedescribed in Nippon Seikagaku Kai (Biochemical Society of Japan) ed.,“Zoku-Seikagaku Jikken Kouza 1, Idenshi Kenkyuho II (Lectures onBiochemical Experiments (Second Series; 1), Methods for Gene Study II)”,p.105 (Susumu HIROSE), Tokyo Kagaku Dojin, Japan (1986); NipponSeikagaku Kai (Biochemical Society of Japan) ed., “Shin-Seikagaku JikkenKouza 2, Kakusan III (Kumikae DNA Gijutsu) (New Lectures on BiochemicalExperiments 2, Nucleic Acids III (Recombinant DNA Technique))”, p.233(Susumu HIROSE), Tokyo Kagaku Dojin, Japan (1992); R. Wu, L. Grossman,ed., “Methods in Enzymology”, Vol.154, p.350 & p.367, Academic Press,New York (1987); R. Wu, L. Grossman, ed., “Methods in Enzymology”,Vol.100, p.457 & p.468, Academic Press, New York (1983); J. A. Wells etal., “Gene”, Vol.34, p.315 (1985); T. Grundstroem et al., “Nucleic AcidsRes.”, Vol.13, p.3305 (1985); J. Taylor et al., “Nucleic Acids Res.”,Vol.13, p.8765 (1985); R. Wu ed., “Methods in Enzymology”, Vol.155,p.568, Academic Press, New York (1987); A. R. Oliphant et al., “Gene”,Vol.44, p.177 (1986), etc., the disclosures of which are herebyincorporated by reference. Examples of such techniques includesite-directed mutagenesis (or site-specific mutagenesis) using syntheticoligonucleotides, Kunkel method, dNTP[α S] method (Eckstein method),area-directed mutagenesis using sulfite, nitrite, etc. and the like.

Further, the proteins thus obtained can be modified chemically for aminoacid residues. The protein can also be modified or partially degradedwith enzymes such as pepsin, chymotrypsin, papain, bromelain,endopeptidase, exopeptidase or the like to produce a derivative. Inaddition, the proteins may be expressed as fusion proteins when they areproduced using gene recombinant techniques, which are subjected to invivo and in vitro conversion into and/or processing to those having abiological activity substantially equivalent to native MT-MMP-3. Thefusion protein production conventionally used in gene engineering can beemployed. Further, such fusion proteins can be isolated and/or purifiedby means of affinity chromatography or the like wherein the techniqueemploys a fusion portion thereof. The structure of proteins can bemodified, improved, etc. by means of methods as described in NipponSeikagaku Kai (Biochemical Society of Japan) ed., “Shin-Seikagaku JikkenKouza 1, Tanpakushitsu VII, Tanpakushitsu Kougaku (New Lectures onBiochemical Experiments 1, Proteins VII, (Protein Engineering))”, TokyoKagaku Dojin, Japan (1993), the disclosures of which are herebyincorporated by reference, or by techniques as described in referencescited therein as well as methods substantially equivalent thereto.

In addition, as described herein below, the biological activity mayinclude those having an immunological activity including an antigenicactivity.

Hence, the present invention relates to proteins wherein one or moreamino acid residues may differ from native amino acid residues from theviewpoint of homology, and proteins wherein the positions of one or moreamino acid residues may differ from those of native residues. Thepresent invention includes deletion analogs wherein one or more aminoacid residues (for example, 1 to 80 residues, preferably 1 to 60residues, more preferably 1 to 40 residues, further more preferably 1 to20 residues, particularly 1 to 10 residues, etc.) characteristic ofMT-MMP-3 are deleted; substitution analogs wherein one or more aminoacid residues (for example, 1 to 80 residues, preferably 1 to 60residues, more preferably 1 to 40 residues, further more preferably 1 to20 residues, particularly 1 to 10 residues, etc.) characteristic ofMT-MMP-3 are replaced with other amino acid residues; and additionanalogs wherein one or more amino acid residues (for example, 1 to 80residues, preferably 1 to 60 residues, more preferably 1 to 40 residues,further more preferably 1 to 20 residues, particularly 1 to 10 residues,etc.) are added. All of the above mentioned variants or the like areincluded in the present invention as long as domain structures orC-terminal transmembrane domains commonly characteristic of MMPs aremaintained. It is thought that MT-MMP-3 of the present invention mayinclude proteins having a primary structural conformation identical withor substantially equivalent to native MT-MMP-3 or a part thereof. It isalso thought that MT-MMP-3 may include proteins having a biologicalactivity identical with or substantially equivalent to native MT-MMP-3.It may be one of mutants (variants) naturally produced or occurred. TheMT-MP-3 according to the present invention can be separated, isolatedand purified as described herein below.

The protein or a salt thereof, which (i) belongs to a member of MMPshaving the capability of activating pro MMP-2, (ii) has an activityidentical with or substantially equivalent to native MT-MMP(particularly, MT-MMP-3), and (iii) is a pro MMP-2 activating factor,excluding MT-MMP-1; or a partial peptide (or peptide fragment) thereofor a salt thereof is useful and valuable in studies on development andresearch of enzyme inhibitors using said protein or the like, researchand development of medicines, studies on biological phenomenon andreaction with which MT-MMP-3 is thought to be associated, etc. Further,the protein and partial peptide or a salt thereof can be used forproduction of antibodies thereagainst. The products of the presentinvention can be used for investigation and research on specific targetsto be assayed or measured.

The present invention also relates to DNA sequences coding for any ofthe above mentioned polypeptides, MT-MMP-3 polypeptides, and MT-MMP-3analogs and derivatives, each having all or part of characteristics,unique properties, etc. of native MT-MMP-3.

The DNA sequences of the present invention provides informationconcerning the amino acid sequences of the mammal proteins that have notbeen known so far. Therefore, utilization of the above information isincluded in the present invention. Such utilization includes design ofany of probes for isolating and detecting mammal, in particular human,genomic DNA or cDNA, encoding MT-MMP-3, related (or associated)proteins, etc.

The DNA sequences of the present invention are useful as probes forisolating and detecting mammal, most preferably human, genomic DNA andcDNA, coding for MT-MMP-3 or related proteins thereof. To isolate genes,PCR techniques or PCR using reverse transcriptase (RT) (RT-PCR) can beused. MT-MMP-3 cDNA and associated DNA thereof can be used in isolatingand detecting MT-MMP-3-related genes, via selecting characteristicsequence regions based on amino acid sequences deduced from the clonedand sequenced MT-MMP-3 cDNA sequence, then designing and chemicallysynthesizing DNA primers, and carrying out PCR, RT-PCR, or any othertechniques with the obtained DNA primers.

Since MT-MMP-3 conserves well the structural characteristics ofMT-MMP-1, there is assumed the possibility that MT-MMP-3 also acts as anactivating factor for pro MMP-2. Therefore, mammal cells such as COS-1cells have been cotransfected with a plasmid for expressing pro MMP-2together with a plasmid for expressing MT-MMP-3. Zymography was carriedout for the recollected culture medium of the cotransfectants. As aresult, a 62 kDa active MMP-2 and a 64 kDa active intermediate have beendetected, other than pro MMP-2 which is primarily observed at theposition of molecule weight 68 kDa, and the activation of pro MMP-2depending on the expression of MT-MMP-3 has been observed.

The expression of MT-MMP-3 mRNA in human tissues has been examined byNorthern blotting for various tissue-derived poly (A) ⁺RNA. As a result,it has been recognized that MT-MMP-3 mRNA is highly expressed in humanlungs, brains, and placentas. However, no expression has been found inhuman hearts, levers, kidneys, spleens, and muscle tissues. In studiesdone by the inventors, the expression of MT-MMP-1 mRNA is significantlyhigh in human lungs, kidneys, and placentas, while lowest in the humanbrains. These observations show that, although MT-MMP-3 is closelyanalogous structurally, and functionally in terms of the capability ofactivating pro MMP-2, to MT-MMP-1, expression of the genes for MT-MMP-3and MT-MMP-1 in the actual tissues is differently regulated. When thecDNA according to the present invention is employed as a probe,techniques including Northern blotting, Southern blotting, in situhybridization or the like enable us to detect and/or measure MT-MMP-3mRNA expression or MT-MMP-3 genes per se in human tissues, which maycontribute greatly to applications to studies on diagnosis and treatmentof tumors (including cancers) such as diagnosis of the presence andabsence of tumor cells, malignancy of cancers, on diagnosis ofAlzheimer's diseases, etc.

According to inventor's investigation results as described herein above,techniques are provided for transferring MT-MMP-3 genes and recombinantDNA molecules into hosts, expressing MT-MMP-3 therein, and isolating andobtaining target MT-MMP-3. Thus, according to the present invention,transformants or transfectants capable of substantially expressingMT-MMP-3 genes and production processes thereof are provided.

In another aspect, the present invention related to nucleic acids, suchas DNA or RNA, which enable us to express

(i) a protein or a salt thereof, (a) being a member of MMPs capable ofactivating pro MMP-2 but not MT-MMP-1, and (b) having an activityidentical with or substantially equivalent to native MT-MMP that is anactivator for pro MMP-2,

(ii) more preferably a polypeptide or a salt (a) having a biologicalproperty or a primary structural conformation, identical with orsubstantially equivalent to MT-MMP-3 or a salt thereof, and (b) havingat least part or all of the protein,

in a prokaryotic cell such as E. coli or an eukaryotic cell such as amammal cell.

In addition, such nucleic acids, particularly DNA, may include (a)sequences coding for an amino acid sequence represented by SEQ ID NO: 2in Sequence Listing or sequences complementary thereto; (b) sequencescapable of hybridizing with the DNA sequences (a) or fragments thereof;and (c) sequences having degenerate codons hybridizable with either ofthe sequences (a) and (b). The unique features of the present inventionalso reside in transformed prokaryotic cells, such as E. coli, andtransformed eukaryotic cells, such as mammal cells, which aretransformed with said nucleic acid and can express the polypeptidesaccording to the present invention.

The present invention further provides antibodies, such as monoclonalantibodies, capable of specifically binding with MT-MMP-3. Theantibodies, such as monoclonal antibodies, of the present inventioncontribute to development and supply of tools useful for researchesassociated with diagnosis of malignant tumors or cancers, as well asstudies on invasion and metastasis of cancers and means useful forresearches associated with the crisis mechanism or diagnostic techniquesAlzheimer's disease. Such tools and means are within the scope of thepresent invention.

The antibody, such as monoclonal antibody, according to the presentinvention can be produced by immunizing animals with, as an immunogen,human MT-MMP-3 obtained according to the present invention based ontechniques known or widely applicable in the art. Examples of suchtechniques are found in Milstein et al., Nature, 256: 495 to 497, 1975,etc., the disclosures of which are hereby incorporated by reference. Inthis technique, the antigen used may include any of naturally-occurring(native) MT-MMP-3, recombinant human MT-MMP-3, synthetic peptides havingan amino acid sequence composed of at least continuous 8 amino acidswhich are part of MT-MMP-3, etc. The monoclonal antibody can be labeledusing conventional techniques. The labels (markers) may include enzymes,prosthetic molecules, pigment (chromophore) substances, fluorescentsubstances, chemiluminescent compounds, photoluminescent substances,radioactive substances or the like.

Described herein below is the production of antibodies.

It goes without saying that the monoclonal antibody to be used in thepresent invention may be a monoclonal antibody obtained by utilizingcell fusion techniques with myeloma cells.

The monoclonal antibody to be used in the present invention can beproduced by the following processes:

1. Preparation of immunogenic antigens (immunogens)

2. Immunization of animals with immunogenic antigens

3. Preparation of myeloma cells

4. Cell fusion between antibody-producing cells and myeloma cells

5. Selection and cloning of hybridomas (hybrid cells)

6. Production of monoclonal antibodies

1. Preparation of immunogenic antigens

The antigen used includes naturally occurring MT-MMP-3 and recombinantMT-MMP-3 as prepared according to the present invention. AlthoughMT-MMP-3 may be used after formation of immunogenic conjugates, it canbe used to immunize animals after being mixed with a suitable adjuvantwithout any modifications. Such antigens can be separated, isolated andpurified from various sources, for example, antigen-producing sourcesincluding cultured cells, cultured tissues, transformant cells, etc. byconventional techniques. Such conventional techniques are, for example,salting out such as ammonium sulfate fractionation,.etc.; gel filtrationon Sephadex™, etc.; ion exchange chromatography using carriers having,for example, a diethylaminoethyl or carboxymethyl group, etc.;hydrophobic chromatography using carriers having, for example, ahydrophobic group such as butyl, octyl, or phenyl, etc.; pigment (orchromophore) gel chromatography; electrophoresis; dialysis;ultrafiltration; affinity chromatography; high performance liquidchromatography; etc. Preferably, the antigen to be used is separated andpurified by polyacrylamide electrophoresis, affinity chromatography inwhich an antibody for specifically recognizing an antigen, such as amonoclonal antibody, is immobilized. Examples of such techniques alsoinclude gelatine-agarose affinity chromatography, heparin-agarosechromatography, etc.

MT-MMP-3 may be fragmented or may include a synthetic polypeptidefragment obtained via selecting specific (or characteristic) sequenceareas based on amino acid sequences deduced from the cloned andsequenced cDNA sequences followed by design and chemical synthesis. Thefragments may be coupled with various carrier proteins via suitablecoupling agents to form immunogenic conjugates such as hapten-proteins.The immunogenic conjugates can be used to design monoclonal antibodiesthat can recognize only specific sequences. A cysteine residue or thelike can be added to the polypeptide thus designed so as to prepare animmunogenic conjugate easily. To fix with a carrier protein or the like,the carrier protein is first activated. This activation may includeincorporation of an activated binding group thereinto, etc. Theactivated binding groups include (1) active ester or active carboxylgroups such as a nitrophenyl ester group, a pentafluorophenyl estergroup, a 1-benzotriazol ester group, and an N-succinimide ester group;(2) active dithio groups such as a 2-pyridyldithio group, etc. Thecarrier proteins include keyhole limpet haemocyanin (KLH), bovine serumalbumin (BSA), ovalbumin, globulin, polypeptides such as polylysine,bacterial components such as BCG or the like.

2. Immunization of animals with immunogenic antigens

Animals can be immunized according to techniques as described in ShigeruMURAMATSU et al. ed., “Jikken Seibutsu Gaku Kouza 14, Men-eki SeibutsuGaku (Lectures on Experimental Biology 14, Immunobiology)”, Maruzen K.K., 1985; Nippon Seikagaku Kai (Biochemical Society of Japan) ed.,“Zoku-Seikagaku Jikken Kouza 5, Men-eki Seikagaku Kenkyuho (Lectures onBiochemical Experiments (Second Series; 5), Methods for Immunologicaland Biochemical Study)”, Tokyo Kagaku Dojin, Japan (1986); NipponSeikagaku Kai (Biochemical Society of Japan) ed., “Shin-Seikagaku JikkenKouza 12, Bunshi Men-eki Gaku III (Kougen-Koutai-Hotai) (New Lectures onBiochemical Experiments 12, Molecular Immunology III(Antigen-Antibody-Complement))”, Tokyo Kagaku Dojin, Japan (1992); etc.,the disclosures of which are hereby incorporated by reference. Theadjuvant to be used with the antigen includes Freund's completeadjuvant, Ribi adjuvant, Bordetella pertussis vaccine, BCG, lipid A,liposome, aluminium hydroxide, silica, etc. Immunization is carried outwith animals, including mice such as BALB/c. The antigen dose is, forexample, approximately 1 to 400 μg/animal for mice. Generally, theantigen is injected intraperitoneally or subcutaneously into a hostanimal, followed by additional immunization by repeated courses whereinintraperitoneal, subcutaneous or intravenous administrations are carriedout approximately 2 to 10 times at 1- to 4-week intervals, preferably 1-to 2-week intervals. For immunization, BALB/c mice, as well as F1 micebetween BALB/c mice and other mice, etc. can be used.

As required, the degree of animal immunization can be assessed byconstructing a system for measuring a titre of antibody and measuringthe titre of an antibody. Furthermore, the present invention relates topolyclonal antibodies against MT-MMP-3 and the production thereof usingrecombinant MT-MMP-3. In this case, the animal used may include mammals,birds or the like. Examples of such animals are cow, horse, goat, sheep,swine, rabbit, mouse, rat, guinea pig, monkey, dog, cat, cock, hen, etc.The antibody may be anti-serum. Also, the antibody may be a higherpurified form. For example, its isolation and purification can becarried out in the same manner as the monoclonal antibody describedherein below.

3. Preparation of myeloma cells

Immortal cell strains (tumor cell lines) to be used for cell fusion canbe selected from non-immunoglobulin-producing cell lines. The cellstrains to be used for cell fusion may include, for example,P3-NS-1-Ag4-1 (NS-1, Eur. J. Immunology, 6, 511 to 519, 1976),SP210-Ag14 (SP2, Nature, 276, 269 to 270, 1978), mouse myeloma MOPC-21cell line-derived P3-X63-Ag8-U1 (P3U1, Current topics in Microbiol. andImmunol., 81, 1 to 7, 1978), P3-X63-Ag8 (x63, Nature, 256, 495 to 497,1975), P3-X63-Ag8-653 (653, J. Immunol., 123, 1548 to 1550, 1979), etc.8-azaguanine resistant mouse myeloma cell lines can be sub-cultured in amedium for cell culture wherein antibiotics such as penicillin, amikacinor the like, fatal calf serum (FCS) or the like and 8-azaguanine (forexample, 5 to 45 μg/ml) are added to a medium for cell culture, such asDulbecco's modified Eagle's medium (DMEM) or RPMI-1640 medium. Thespecified number of cell lines can be prepared by passing the normalmedium two or five days before cell fusion. The cell lines to be usedmay be cultured on the normal medium after the frozen and preservedstrains have been completely thawed at approximately 37° C. and havebeen washed on the normal medium such as RPMI-1640 three or more times,and the specified number of cell strains may be prepared.

4. Cell fusion between antibody-producing cells and myeloma cells

After animals such as mice are immunized according to the above step 2,their spleens are removed in two to five days from final immunization,and the spleen cell suspension is obtained. In addition to the spleencells, lymph node cells at various sites of organisms can be obtainedand used for cell fusion. The spleen cell suspension thus obtained andthe myeloma cell strains obtained by the above step 3 are placed in amedium such as minimum essential medium (MEM), DMEM or RPMI-1640 medium,and an agent for cell fusion, fusogen, such as polyethylene glycol, isadded. A widely-used agent for cell fusion can be used, including HVJ:Hemagglutinating virus of Japan (Sendai virus). Preferably, 0.5 to 2 mlof 30 to 60% polyethylene glycol can be added. Polyethylene glycol with1,000 to 8,000 in molecule weight can be employed, more preferably,polyethylene glycol between 1,000 and 4,000 in molecule weight. Thepreferred concentration of polyethylene glycol in the fusion medium isbetween 30 and 60%. As required, a small amount of dimethyl sulfoxide orthe like is added to promote fusion. The ratio of spleen cells(lymphocytes):myeloma cell lines to be used for fusion is preferably 1:1to 20:1, and preferably falls between 4:1 and 7:1.

The fusion reaction is conducted for one to 10 minutes, before theaddition of a medium such as RPMI-1640 medium. Fusion reactionprocessing can be done several times. After fusion reaction processing,cells are separated by a centrifuge, then transferred to the selectionmedium.

5. Selection and cloning of hybridomas (hybrid cells)

The selection media include conventionally known “HAT medium”, i.e.,FCS-containing MEM, RPMI-1640 medium, etc., supplemented withhypoxanthine, aminopterin, and thymidine. The replacement method for theselection medium is to replenish an amount equivalent to the capacitydispensed to the medium plate on the following day, after which themedium is replaced by half an amount in HAT medium every one to threedays. The replacement can be modified depending on situations. Eight tosixteen days after fusion, the medium may be replaced every one to fourdays with conventionally known “HT medium” wherein aminopterin isexcluded from HAT medium. As a feeder cell, for example, mouse thymocytecan be used, which is sometimes effective.

The supernatant of the culture well with highly growing hybridoma isscreened by using MT-MMP-3 or a peptide fragment thereof as an antigenor by using a labeled anti-mouse antibody for measuring targetantibodies, with a measuring system such as radioimmunoassay (RIA),enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay(FIA) or by the fluorescence activated cell sorter (FACS), etc. Thetarget antibody-producing hybridoma is cloned. Cloning is carried out bypicking up colonies in the agar medium or preferably by the limitingdilution. Cloning should be performed several times.

6. Production of monoclonal antibodies

The obtained hybridoma cells are cultured in a suitable growth mediumsuch as FCS-containing MEM, RPMI-1640 medium or the like, and a desiredmonoclonal antibody can be obtained from the culture supernatant. Largeamounts of monoclonal antibodies can be produced by propagatinghybridomas as ascites tumors. In this case, each hybridoma is implantedintraperitoneally in a histocompatible animal isogenic to an animal fromwhich the myeloma cell is derived and is propagated. Or each hybridomacan be inoculated, for example, in nude mice, and propagated to producethe monoclonal antibody in the ascites of the animals. The producedmonoclonal antibody can be collected from the ascetic fluid andobtained. Prior to implantation of hybridomas, the animal is pretreatedintraperitoneally with mineral oils such as pristane(2,6,10,14-tetramethylpentadecane). After the preconditioning, thehybridoma can be propagated therein and the ascitic fluid can beharvested. The ascitic fluid can be used as a monoclonal antibodywithout purification or after purification by conventionally knownmethods, including salting out such as precipitation with ammoniumsulfate, gel filtration with Sephadex, ion exchange chromatography,electrophoresis, dialysis, ultrafiltration, affinity chromatography,high-performance liquid chromatography, and can be employed. Preferably,the monoclonal antibody-containing ascitic fluid is fractionated withammonium sulfate and separated and purified by treatments with cationicion exchange gel such as DEAE-Sepharose, an affinity column such asprotein A column, etc. More preferably, it is treated with affinitychromatography with immobilized antigens or antigen fragments (forexample, synthetic peptides, recombinant antigen proteins or peptides,portions capable of specifically recognizing the antibody); affinitychromatography with immobilized protein A; etc.

It is possible to produce antibodies by recombinant DNA techniqueswherein the antibody thus obtained in a large amount is sequenced and/ora nucleotide sequence coding for the antibody obtained from thehybridoma cell line is employed.

These antibodies may be treated with enzymes such as trypsin, papain,pepsin or the like to produce antibody fragments including Fab, Fab′,and F (ab′)₂ that are occasionally obtained by reduction. These antibodyfragments may be occasionally used.

The antibody to be labeled with a marker may include IgG fractions, andspecific bonding fragments Fab′ obtainable by reduction after pepsindigestion. The labels include enzymes (peroxidase, alkaline phosphatase,or β-D-galactosidase or the like), chemical substances, fluorescences,radioisotopes, or the like.

In the present invention, detection and measurement can be carried outby immunostaining including, for example, staining of tissues and cells,immunoassays including, for example, competitive immunoassay andnon-competitive immunoassay, radioimmunoassay, ELISA, or the like. Thedetection and measurement can also be carried with or without B-Fseparation. Preferably, the detection and measurement is carried out bymeans of radioimmunoassay, enzyme immunoassay or sandwich assay. In thesandwich-type assay, one of the antibody pair against MT-MMP-3 isdetectably labeled. The other antibody capable of recognizing the sameantigen is immobilized on a solid phase. Incubation is carried out tosequentially react a sample to be assayed, labeled antibodies, andimmobilized antibodies as required. After the non-binding antibodies areseparated, the label or marker is detected or measured. The amount ofthe measured label is proportional to the amount of antigen, i.e.,MT-MMP-3. For this assay, simultaneous sandwich assay, forward sandwichassay, or reverse-sandwich assay or the like is called according to theaddition sequence of the insolubilized antibody and the labeledantibody. For example, washing, stirring, shaking, filtration,pre-extraction for antigen, etc. is optionally adopted in themeasurement process under specific conditions. The other measurementconditions such as specific regents, concentration of bufferingsolution, temperature or incubation time can vary according to theelements, such as concentration of the antigens in the sample or thenature of samples to be measured. Any person ordinary skilled in the artcan suitably select and determine optimal conditions effective for eachmeasurement while using the general experimentation and perform theselected measurement.

Various carriers capable of immobilizing antigens or antibodies areavailable in the art, and they can be arbitrarily and suitably selectedin the present invention. For the carrier, various carriers which can beused for antigen and antibody reactions are known. It goes withoutsaying that any well-known carrier can be selected and used in thepresent invention. Preferred examples are inorganic materials including,for example, glass such as activated glass and porous glass, silica gel,silica-alumina, alumina, magnetized iron, magnetized alloy, etc.;organic high molecular substances including, for example, polyethylene,polypropylene, polyvinyl chloride, polyvinylidene fluoride, polyvinylacetate, polymethacrylate, polystyrene, styrenelbutadiene copolymer,polyacrylamide, cross-linked polyacrylamide, styrene/methacrylatecopolymer, polyglycidyl methacrylate, acrolein/ethylene glycoldimethacrylate copolymer, etc., cross-linked albumin, collagen, gelatin,dextran, agarose, cross-linked agarose, natural or modified cellulosesuch as cellulose, microcrystalline cellulose, carboxymethylcellulose,cellulose acetate and the like, cross-linked dextran, polyamide such asnylon, polyurethane, polyepoxy resin and the like; those obtained byemulsifying polymerization thereof; cells, erythrocytes and the like;and those into which a functional group may be introduced, as required,by using a silane coupling agent.

Also included are solid materials such as filtration paper, beads, innerwall of test container such as test tube, titer plates, titer wells,glass cells, cells made of synthetic materials such as plastic resincells, glass rods, rods made of synthetic materials, rods thickened orthinned at the end, rods whose end is round or flat, and thin-platedrods.

Antibodies can be coupled with these carriers, and preferably themonoclonal antibodies according to the present invention which arecapable of specifically binding with MT-MMP-3, can be coupled therewith.Coupling between the carrier and those associated with theseantigen-antibody reactions can be carried out by techniques includingphysical method such as adsorption; a chemical method using a couplingagent, etc. or an activated reactant; a method using a chemicallyinteractional coupling.

The label may include enzyme, enzyme substrates, enzyme inhibitors,prosthetic groups, coenzymes, enzyme precursors, apoenzymes, fluorescentsubstances, pigments, chemical luminescent compounds, light-emittingsubstances, coloring substances, magnetic substances, metal particlessuch as gold colloids, radioactive substances and the like.

The enzyme may include oxidation-reduction enzymes such asdehydrogenase, reductase, and oxidase; transferases that catalyze thetransfer of an amino, carboxyl, methyl, acyl, phosphate group or thelike; hydrolases that hydrolyze an ester, glycoside, ether, peptide bondor the like; lyase; isomerase; ligase; and the like. Plural enzymes canbe used in a conjugated form for detection (for example, enzymaticcycling may also be utilizable).

Typical radioactive isotopes for the label include [³²P], [¹²⁵I],[¹³¹I], [³H], [¹⁴C], [³⁵S], and the like.

Typical enzymes for the label include peroxidases such as horseradishperoxidase; galactosidase such as E. coli β-D-galacosidase; maleatedehydrogenase; glucose-6-phosphate dehydrogenase; glucose oxidase;gluocoamylase; acetylcholine esterase; catalase; alkaline phosphatasesuch as calf intestinal alkaline phosphatase and E. coli alkalinephosphatase, and the like.

In the case where alkaline phosphatase is used, fluorescence or emittedlight can be measured by using a substrate such as umbelliferonederivatives including 4-methylumbellipheryl phosphate; phenol phosphatederivatives including nitrophenyl phosphate; enzymatic cycling systemsutilizing NADP; luciferin derivatives; dioxetane derivatives; and thelike. It is also possible to use a luciferin/luciferase system.

When catalase is used, the reaction takes place with hydrogen peroxideto produce oxygen which can be detected with an electrode or the like.The electrode may be a glass electrode, an ionic electrode using aninsoluble salt membrane, a liquid-membrane type electrode, a polymermembrane electrode and the like.

It is possible to replace the enzyme label with a biotin label and anenzyme-labeled avidin (streptoavidin)

For the label, a plurality of many different kinds of labels or markerscan be used. In this case, it is possible to perform plural measurementscontinuously or discontinuously and/or simultaneously or separately.

According to the present invention, a signal can be formed by using acombination of 4-hydroxyphenylacetic acid, 1,2-phenylenediamine,tetramethylbenzidine, or the like with horseradish peroxidase, by usinga combination of umbelliferyl galactoside, nitrophenyl galactoside, orthe like with enzyme reagents such as β-D-galactosidase andglucose-6-phosphoric acid dehydrogenase. There can be further used thosethat are capable of forming a quinol compound such as hydroquinone,hydroxybenzoquinone or hydroxyanthraquinone, a thiol compound such aslipoic acid or glutathione, phenol derivatives or ferrocene derivativesby utilizing the action of enzymes.

The fluorescent substance and chemiluminescent compounds may includefluorescein isothiocyanate, Rhodamine derivatives such as Rhodamine Bisothiocyanate, and tetramethyl Rhodamine isothiocyanate, dansylchloride (5-(dimethylamino)-1-naphtalenesulfonyl chloride), dansylfluoride, fluorescamine(4-phenylspiro[furan-2(3H),1′-(3′H)-isobenzofuran]-3,3′-dione),phycobiliprotein, acridinium salts, luminol compounds such as lumiferin,luciferase, and aequorin, imidazole, oxalic acid ester, rare earthchelate compounds, cumarin derivatives, etc.

The labelling can be accomplished by utilizing the reaction of a thiolgroup with a maleimide group, reaction of a pyridyldisulfide group witha thiol group, the reaction of an amino group with an aldehyde group,etc. Additionally, it can be selected from widely known methods, methodsthat can be easily put into practice by an artisan skilled in the art,or any of methods modified therefrom. The coupling agents used forproducing the foregoing immunoconjugate or for coupling with carriersare also applicable and usable.

The coupling agents include, for example, glutaraldehyde, hexamethylenediisocyanate, hexamethylene diisothiocyanate, N,N′-polymethylenebisiodoacetamide, N,N′-ethylene bismaleimide, ethylene glycolbissuccinimidyl succinate, bisdiazobenzidine,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, succinimidyl3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl4-(N-maleimidometyl)cyclohexane-1-carboxylate (SMCC),N-sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate,N-succinimidyl (4-iodoacetyl)-aminobenzoate, N-succinimidyl4-(1-maleimidophenyl)butyrate, N-(ε-maleimidocaproyloxy)succinimide(EMCS), iminothiolane, S-acetylmercaptosuccinic anhydride,methyl-3-(4′-dithiopyridyl)propionimidate,methyl-4-mercapto-butyrylimidate, methyl-3-mercaptopropionimidate,N-succinimidyl-S-acetylmercaptoacetate, etc.

According to the measurement of the present invention, substances to bemeasured can be made to react sequentially with labeled antibodyreagents such as monoclonal antibodies labeled with enzymes or the like,and with antibodies coupled (immobilized) on a carrier, or all themembers can be reacted each other simultaneously. The sequence of addingreagents (members) may vary depending on the type of carrier systemselected. In the case where beads such as sensitized plastics are used,the labeled antibody regents such as monoclonal antibodies labeled withenzymes or the like are first put in a suitable test tube, together witha sample including substances to be measured, followed by addition ofthe plastic beads. Measurement can be then carried out.

For quantitative measurements according to the present invention, theimmunological measurement is applied. For the measurement, the solidphase carriers used may include various materials and shapes which canbe selected from balls, microplates, sticks, microparticles, test tubes,and the like, made of polystyrene, polycarbonate, polypropylene,polyvinyl and other materials capable of adsorbing proteins such asantibodies.

The measurement can be carried out in a suitable buffer system so as tomaintain an optimal pH (for example, between pH about 4 and about 9). Inparticular, the preferred buffers may include acetate buffer, citratebuffer, phosphate buffer, tris buffer, triethanolamine buffer, boratebuffer, glycine buffer, carbonate buffer, tris-hydrochloride buffer,etc. The buffers can be used optionally in a mixed form at an arbitraryrate. Preferably, the antibody and antigen reaction is carried out at atemperature between about 0 and 6 ° C.

The antibody regents (for example, monoclonal antibodies labeled withenzymes), the regents such as antibodies immobilized on (coupled to) acarrier, and substances (samples) to be measured can be incubated untilequilibrium is reached. However, the reaction can be stopped afterlimited incubation by separating the solid phase from the liquid phaseat a time well before the antibody/antigen equilibrates, and the degreeof the presence of markers such as enzymes in either of the liquid andsolid phases can be measured. Measurement operation can be performed byusing automated measuring instruments, and data can be measured bypermitting a substrate to be converted by the action of enzymes and bydetecting produced indication signals with a luminescence detector, aphoto detector or the like.

In the antibody/antigen reaction, adequate means can be taken so as tostabilize regents to be used, substances (samples) to be measured, andlabels (markers) such as enzymes, respectively, and/or to stabilizeantibody/antigen reactions per se. Further, for eliminating non-specificreaction, reducing inhibitory influences acting thereon, and/oractivating measurement reaction, proteins, stabilizers, surfactants,chelating agents or the like can be added to solutions which areincubated. The chelating agent is more preferably ethylenediaminetetraacetate. The blocking techniques for preventing non-specificbinding reaction, which techniques are generally employed in the art orwell-known among the persons skilled in the art, may be employed. Theblocking can be achieved by treatments with normal serum proteins,albumin, skim milk or the like from mammals, etc., fermented milkproducts, collagen, gelatin, or the like. These methods or techniquescan be used without any limitation since the purpose is to preventnon-specific binding reaction.

The samples to be measured according to the present invention mayinclude various types of solutions such as colloid solution, non-fluidsamples and the like. Preferably, the samples are biological samplesincluding, for example, blood, serum, plasma, articular fluid,cerebrospinal fluid, saliva, amniotic fluid, urine, any other humoralfluids, cell culture liquids, tissue culture liquids, tissue homogenate,biopsy samples, tissues, cells and the like.

It should be understood that the DNA of the present invention can betreated in the similar manner as the foregoing antibodies (for example,the DNA can be labeled by well-known techniques or substantiallyequivalents thereto, and can be used for measurements or assays).

By utilizing the foregoing various preferred embodiments according tothe present invention, there can be provided diagnostic means useful forresearches regarding diagnosis or therapy of cancers (malignant tumors),including diagnosis of the presence or absence of tumor cells,estimation of malignancy of cancers and tumors as well as a variety oftechnological means to be applied to the other medical and physiologicalapplications.

By referring to the working examples, the present invention is describedbelow in detail. It should be understood that the present invention isnot limited to such examples and a variety of preferred embodimentswithin the spirit of this specification are enabled.

In the case where nucleotides (bases), amino acids or the like areindicated by abbreviations in the specification and in the drawings,they must conform with an “IUPAC-IUB Commission on BiochemicalNomenclature” or are based on the meanings of the terms which arecommonly used in the art. When optical isomers are present in aminoacids, an L-isomer is referred to unless otherwise specified.

The transformant Escherichia coli, designated NM533 XL1-Blue(XL1-Blue/MMP-X2), obtained in Example 1 (e) mentioned herein below hasbeen deposited as from Jul. 5, 1995 (original deposit date) with theNational Institute of Bioscience and Human Technology (NIBH), Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry, Japan, located at 1-3, Higashi 1-chome, Tsukuba-shi, IBARAKI(Zip Code: 305), JAPAN and has been assigned the Accession Number FERMP-15033. The original deposit of the transformant E. coli NM533 XL1-Blue(XL1-Blue/MMP-X2) has been transferred to one under the Budapest Treatyby a request dated Jul. 1, 1996 and is on deposit with the AccessionNumber FERM BP-5573 under the terms of the Budapest Treaty at NIBH.

The mouse-derived monoclonal anti-human membrane-type matrixmetalloproteinase-3 (MT-MMP-3) antibody producing hybridoma, designated117-4E1, obtained in Example 3 (f) to (h) mentioned herein below hasbeen deposited as from Jul. 5, 1995 (original deposit date) with NIBHand has been assigned the Accession Number FERM P-15031. The originaldeposit of the hybridoma 117-4E1 has been transferred to one under theBudapest Treaty by a request dated Jul. 1, 1996 and is on deposit withthe Accession Number FERM BP-5572 under the terms of the Budapest Treatyat NIBH.

EXAMPLES

Described below are examples of the present invention which are providedonly for illustrative purposes, and not to limit the scope of thepresent invention. In light of the present disclosure, numerousembodiments within the scope of the claims will be apparent to those ofordinary skill in the art.

Example 1

Isolation of Novel Metalloproteinase (MT-MMP-3) cDNA

Isolation of novel MMP cDNA is basically carried out according to thefollowing methods:

1) Degenerate primers were synthesized based on the conservativesequences in an MMP family. Screening for cDNA derived from humantissues was carried out, and PCR products were obtained. 2) The obtainedpartial clones were used as probes, and full length cDNA was screenedfrom cDNA libraries.

(a) Construction of cDNA Libraries

Total RNA extracts from various human tissues (placenta, oral cancers,lung cancers or the like) or cultured cells (human fibrosarcoma cellHT1080, human monocytic leukemia cell U937 or the like) can be used asRNA sources in producing cDNA libraries. mRNA samples derived from anoral malignant melanoma were used as starting materials in Example 1.

Extraction of total RNA from the tissues was carried out according tothe guanidine-cesium chloride technique (Biochemistry, 18: 5294 to 5299,1979), and the total RNAs thus obtained was purified using oligo(dT)cellulose column. cDNA was synthesized according to the Gubler &Hoffman's method (Gene, 25: 263 to 269, 1983). The purified poly (A)mRNA as a template was treated with SuperScript™ reverse transcriptase(Stratagene) using, as primers, random hexamers or oligo dT tosynthesize first-strand cDNA. The first strand cDNA product was treatedwith RNase H, followed by treatment with E. coli DNA polymerase I,whereby second strand cDNA was synthesized to form double-stranded cDNA.For the synthesis of first strand cDNA, a mixture of 5 μl of poly A⁺mRNAfraction samples, 2 μl of random hexamers (80 μM), and 4.5 μl ofreaction buffer solution was incubated for 10 minutes at 70° C. andice-cooled. To the reaction mixture were added 4 μl of 5× reactionbuffer solution, 2 μl of 0.1M dithiothreitol (DDT), 1 μl of 10 mM dNTPsand 1 μl of RNase inhibitor. The mixture was well mixed, to which 0.5 μl(approximately 100 units) of SuperScript™ reverse transcriptase (GIBCOBRL) was added. The resultant mixture was incubated for one hour at 37°C., and then for 10 minutes at 70° C. The synthesis of the second chainof cDNA can be similarly processed and carried out.

Construction of cDNA libraries can be carried out, for example, using λgt11. The synthesized double strand cDNA was blunted with T4 DNApolymerase, followed by methylation of EcoRI site existing in the cDNAwith EcoRI methylase. The cDNA was ligated with EcoRI linkerd(pGGAATTCC) by T₄ DNA ligase, and digested with EcoRI to construct cDNAhaving both EcoRI ends. The resulting cDNA was cloned into EcoRI site ofλ gt11. Then, the cDNA was packaged by an in vitro packaging kit, andcDNA libraries were constructed. A variety of commercially availablehuman tissue-derived cDNA libraries (CLONTECH) can be used directlyherein.

(b) Amplification of Novel MMP cDNA Fragments

A polymerase chain reaction (PCR) with Taq DNA polymerase was carriedout using the obtained cDNA as a template and degenerate primerssynthesized based on the amino acid sequences conserved in the MMPfamily. PCR amplification of novel cDNA fragments was done using, forexample, methods as described in R. Saiki, et al., Science, Vol. 230,pp, 1350 (1985); R. Saiki, et al., Science, Vol. 239, pp. 487 (1985);PCR Technology, Stockton Press, etc.

One μ of the reaction product obtained in the above process as atemplate, 5 μl of 10× PCR buffer solution, 1 μl of 25 mM dNTPs, 1 μl ofprimers for amplification, and 1 unit of Taq polymerase was mixedtogether with sterile distilled water such that the total amount was 50μ 1. This reaction mixture was subjected to PCR amplification with 30cycles wherein one cycle includes 93° C. for one minute, 55° C. for oneminute, and 72° C. for one minute.

The degenerate primers were designed and synthesized as follows:

GEADIMI (SEQ ID NO: 11) (corresponding to Gly¹⁵⁵ to Ile¹⁶¹ of MMP-1,Gly¹⁶⁵ to Ile¹⁷¹ of MMP-2, Gly¹⁵⁵ to Ile¹⁶¹ of MMP-3, Gly¹⁵⁰ to Ile¹⁵⁶of MMP-7, Gly¹⁵⁴ to Ile¹⁶⁰ of MMP-8, Arg¹⁶² to Ile¹⁶⁸ of MMP-9, Gly¹⁵⁴to Ile¹⁶⁰ of MMP-10, Gly¹⁵¹ to Ile¹⁵⁷ of MMP-11, and Gly¹⁵⁵ to Val¹⁶¹ ofMMP-12, respectively; numbering of amino acid residues is according toFIGS. 1A to 1E), and GDAHFDDDE (SEQ ID NO: 10) (corresponding to Gly¹⁹²to Glu²⁰¹ of MMP-1, Gly²⁰³ to Glu²¹¹ of MMP-2, Asn¹⁹² to Glu²⁰¹ ofMMP-3, Gly¹⁸⁷ to Glu¹⁹⁶ of MMP-7, Gly¹⁹¹ to Glu²⁰⁰ of MMP-8, Gln¹⁹⁹ toGlu²⁰⁸ of MMP-9, Tyr¹⁹¹ to Glu²⁰⁰ of MMP-10, Glu¹⁸⁸ to Glu¹⁹⁷ of MMP-11,and Gly¹⁹² to Glu²⁰¹ of MMP-12, respectively; numbering of amino acidresidues is according to FIGS. 1A to 1E) were selected as well conservedamino acid sequences from the catalytic domains among the known MMPfamily members (each amino acid residue in the sequences served asprimers is represented by a standard single character symbol). Based onabove amino acid sequences, the degenerate oligonucleotide primers for5′-primer (5′-primer: 5P-4) of the following sequence:

(SEQ ID NO: 3)

5′-(C or G)G(A, C, G or T)(A, C or G)(A, C or G)(A, C, G or T)GC(A orT)GA(C or T)AT(A or C)(A or G)T(C or G)AT-3′

and 3′-primer (3′-primer: 3P-2) of the following sequence:

(SEQ ID NO: 4)

5′-(C or T)TC(A or G)T(C or G)(A, C, G or T)TC(A or G)TC(A or G)AA(A orG)TG(A or G)(A or G)(A, C or T)(A or G)TC(C or T)CC

were synthesized according to β-cyanoethylphosphoamidite techniques witha DNA synthesizer: Model 392 (Applied Biosystems).

In the above-mentioned sequences, the parentheses indicate theincorporation of a plurality of bases, leading to multipleoligonucleotides in the primer preparation. The plural bases inparentheses were incorporated in the presence of a mixture of pluralbases upon synthesis.

Upon the synthesis, a BamHI site was introduced onto the 5′-end of theprimer: 5P-4, and an EcoRI site onto the 3′-end of the primer: 3P-2. Theobtained primers 5P-4 and 3P-2 were purified with a Nick column(Pharmacia) equilibrated with a 10 mM sodium phosphate buffer solution(pH 6.8). Absorption at 260 nm was measured and the primer solution wasmade to 20 μM.

The obtained PCR products were separated with 10% agaroseelectrophoresis. Seven types of PCR products with predicted sizes (90 to120 base pairs) from a set of the primers used were extracted andpurified. Each purified PCR product was treated with BamHI and EcoRI,followed by subcloning into, for example, the BamHI, EcoRI site of asuitable plasmid such as pBluescript™ or pUC18. For example, 10 μl ofPCR products were separated and confirmed by means of 10% polyacrylamidegel electrophoresis (PAGE), and approximately 120 to 130 bp PCR productswere subcloned into a plasmid pBluescript™ vector. A reaction mixture of1 μl of PCR products, 1 μl of 10× ligation buffer solution, 2 μl ofresuspended vector solution and 1 μl of T4 DNA ligase were incubated at12° C. overnight. The obtained recombinant vector was introduced intosuitable competent cells (for example, competent E. coli HB101 andcompetent XL1-Blue can be used) and subcloned in accordance with theprotocols of a TA Cloning Kit (Invitrogen). Additionally, vectors suchas pUC119 and pCR™ can be used. The nucleotide sequences of the clonedPCR products were sequenced using a fluorescent DNA sequencer Model 373A(Applied Biosystems) and a Taq dyeprimer cycle sequencing kit (AppliedBiosystems).

The nucleotide sequences of these seven isolated and sequenced PCRproducts were compared with the nucleotide sequences of the known MMPmembers. As a result, two of the seven cloned cDNA fragments matched aportion of the already reported nucleotide sequence of MMP-2 (J. Biol.Chem., 261: 6600 to 6605, 1986) and the one matched a part of thenucleotide sequence of MMP-9 (J. Biol. Chem., 264: 17213 to 17221,1989). Among four other PCR products, two cloned cDNA fragments were thenucleotide sequence irrelevant to MMPs; however, the remaining two were93 bp, have the same sequence each other and conserve the deducedamino-acid sequence showing homology to the reported MMP genes. Forconvenience, this PCR product was named “MMP-X2 fragment”.

(c) Screening of Novel MT-MMP-3 Genes from cDNA Library and Sequencing

Twenty-five ng of MMP-X2 fragments (cDNA fragments) obtained in theforegoing (b) were labeled with [α-³²P] dCTP (Amersham), for example,using a random primed DNA labeling kit (Boehringer Mannheim), therebythe obtained probe having a specific activity of 2 to 5.0 CPM/μg. Thiswas used as a probe for screening for cDNA libraries derived fromvarious human tissues and cells.

Host cells, E. coli Y1090, were infected with human oral malignantmelanoma cDNA libraries constructed in λ gt11 as described in the above(a) at a concentration of 4×10⁴ plaque forming units/15 cm² plate toform plaques. The E. coli Y1090 cell was first cultured in a L mediumcontaining 0.02% maltose overnight, collected, and then suspended in 10mM MgSO₄. This cell suspension was mixed with a phage solution and theresultant mixture was incubated at 37° C. for 15 min. to allow theadsorption of phages on host cells. The infected cells were spread on 15cm² L plates prepared in advance by addition of soft agar. The plateswere incubated at 42° C. overnight to form plaques. Then a nylon filter(for example, Hybond-N: Amersham, etc.) or nitrocellulose filter (forexample, HATF: Millipore, etc.) was placed on the plate and was allowedto stand for about 30 seconds. The membrane (filter) was gently removedand dipped in an alkali denaturing solution (0.5M NaOH and 1.5M NaCl)for 1 min., and then in a neutralizing solution (0.5M Tris-HCl buffer(pH 8) containing 1.5M NaCl) for 15 min. This filter was washed with 2×SSPE (0.36M NaCl, 20 mM NaH₂PO₄, and 2 mM EDTA), and then was air-dried.Transfer of the plaques to filters was repeated, and at least twofilters were replicated. However, the contact time for the second andsubsequent filters with the plates was extended to approximately 2minutes.

These filters were baked at 80° C. for two hours to fix DNAs thereon. Atleast two filters prepared from a single plate were rinsed with awashing solution (50 mM Tris-HCl buffer (pH 8.0) containing 1M NaCl, 1mM EDTA, and 0.1% sodium dodecylsulfate (SDS)) at 42° C. for an hour,respectively, then placed into a bag for hybridization and dipped in aprehybridization solution (50% formamide, 5× Denhardt's solution (0.2%bovine serum albumin, 0.2% polyvinylpyrolidone), 5× SSPE, 0.1% SDS, 100μg/ml thermally-denatured salmon sperm DNA), followed byprehybridization at 42° C. for 6 to 8 hours. Next, to theprehybridization solution was added the ³²P-labeled probe described inthe above (c) which was thermally denatured at 100° C. for 5 min., andhybridization was carried out at 42° C. overnight. After completion ofhybridization, the filters were rinsed in a large amount of 2× SSCsolution containing 0.1% SDS at room temperature. Next, the filters wereplaced in a 0.2× SSC solution containing 0.1% SDS at 55° C. for 30 min.After this treatment was repeated twice, the filters were air-dried.Then each filter was put on an X-ray film (Kodak XR) and autoradiographywas carried out at −80° C. for 12 hours. The X-ray films were developed.Two films obtained from a single plate were superposed, and overlappingsignals were marked. Plaques corresponding to the marked signals werepicked up and suspended in an SM solution (50 mM Tris-HCl buffer (pH7.5) containing 100 mM NaCl and 10 mM MgSO₄). This phage suspension wassuitably diluted, preferably diluted at a concentration of 10 to 100plaque forming units/10 cm² plate, and plated on 10 cm² plates on whichE. coli was cultured. Then screening was carried out in the same manneras above, and recombinant phages were obtained.

(d) Preparation of Recombinant λ gt11 DNA Having Novel MT-MMP-3 Gene

The cloned phages were plated respectively in the same manner as thatdescribed in the foregoing (c), and incubated at 42° C. for 3 hours, andthen at 37° C. overnight. To the SM solution were added several drops ofchloroform, and the plates were allowed to stand at room temperature for30 min. A plug of soft agar in the upper layer was obtained byscratching together with the SM solution, followed by centrifugation. Tothe centrifuged supernatant was added polyethylene glycol-6000(PEG-6000) until a final concentration of 10% was reached, the mixturewas stirred, and allowed to stand at 4° C. for 1 hour. This wascentrifuged, the supernatant was discarded, and phage particles wererecollected. The phage particles were suspended in a SM solution andpurified by glycerol-gradient ultra centrifugation (Molecular cloning, alaboratory manual, Ed. T. Maniatis, Cold Spring Harbour Laboratory, 2ndEd. 78, 1989). The obtained phages were suspended in a TM solution, andtreated with DNase I and RNase A, to which then was added a mixture of20 mM EDTA, 50 μg/ml Proteinase K, and 0.5% SDS. The mixture wasincubated for 1 hour at 65° C. The resultant mixture was extracted withphenol, then with diethyl ether, and precipitated with ethanol to affordDNA. The obtained DNA was washed with 70% ethanol, dried, and dissolvedin a TE solution (10 mM Tris-HCl buffer (pH 8) containing 10 mM EDTA).

(e) Sequencing of Inserts

The λ gt11 DNA prepared in the foregoing (d) was cleaved with EcoRI. Theinserts were separated and purified, then subcloned into the EcoRI siteof a vector pBluescript™ (Stratagene). Host cells, E. coli NM533XL1-Blue, were transformed with this recombinant pBluescript. After F′selection of the transformed cells, the cells were infected with helperphages, VCSM13 (Stratagene), and cultured overnight. The cultured mediumwas centrifuged to remove bacterial cells, and PEG/NaCl was added tothis medium to precipitate phages. The precipitate was suspended in a TEsolution, then extracted with phenol and precipitated with ethanol torecover single strand DNAs. The single strand DNA was sequenced using afluorescent DNA sequencer Model 373A (Applied Biosystems) and a Taqdyeprimer cycle sequencing kit (Applied Biosystems). The sequencedfull-length nucleotide sequence was 2116 base pairs, and described inSEQ ID NO: 1 of the Sequence Listing. For the nucleotide sequence of SEQID NO: 1, a matching sequence was checked using GENBANK/EMBL DNA DataBase; however, there exists no same sequence. It has been recognizedthat an open reading frame potentially encoding a putative 607-aminoacid protein is present in this approximately 2.1-kilobase pair DNAsequence, of which amino acid sequence is shown in SEQ ID NO: 2 in theSequence Listing. The deduced protein has been named “MT-MMP-3”. Theobtained DNA fragments can be incorporated into vectors includingplasmids, such as PEX, pMEMneo, or pKG and can be expressed in hostcells such as E. coli or CHO cells.

The Escherichia coli, designated NM533 XL1-Blue (XL1-Blue/MMP-X2),harboring a vector (pSG5 ™ (Stratagene)) into which a nucleotidesequence coding for the above MT-MMP-3 is incorporated has beendeposited as from Jul. 5, 1995 (original deposit date) with NIBH and hasbeen assigned the Accession Number FERM P-15033. The original deposit ofthe transformant E. coli NM533 XL1-Blue (XL1-Blue/MMP-X2) has beentransferred to one under the Budapest Treaty by a request dated Jul. 1,1996 and is on deposit with the Accession Number FERM BP-5573 under theterms of the Budapest Treaty at NIBH.

(f) Amino Acid Sequence Analysis of MT-MMP-3

FIGS. 1A to 1E show an alignment when the amino acid sequence (describedin SEQ ID NO: 2 in Sequence Listing) deduced from the MT-MMP-3nucleotide sequence (described in SEQ ID NO: 1 in Sequence Listing) iscompared with the known amino acid sequences of MMP members. The aminoacid sequence as shown in SEQ ID NO: 2 in Sequence Listing shows highhomology to the MMP family, and conserves well characteristic domainstructures of the MMP family, i.e., a signal peptide removable duringsecretion and production, a propeptide domain, a catalytic domain, ahinge domain, and a hemopexin-like domain. In particular, PRCGVPD (SEQID NO: 12), which is the most conservative sequence among the MMP familymembers and positioned at or near a cleavage site for converting a proform into an active form, is conserved completely in MT-MMP-3, and thesequence of an active domain is also highly conservative. Comparison ofMT-MMP-3 with the other known MMP family members for the amino acidsequences of the active domain including a Zn²⁺-binding site, revealsthat the homology of MT-MMP-3 protein is the highest to MT-MMP-1 (68%)and significantly to others, including MMP-12 (49%), MMP-2 (52%) MMP-9(48%), MMP-1 (49%), MMP-3 (48%), MMP-8 (49%), MMP-11 (42%), and MMP-7(44%).

In addition, MT-MMP-3 has three characteristic insertions compared withthe other MMP family members. They are the 11-amino acid insertion,GSSKFHIRRKR (IS-1: Gly¹⁰⁹ to Arg¹¹⁹ of SEQ ID NO: 2), between apropeptide domain and a catalytic domain, the 8-amino acid insertion,PYSELENG (IS-2: Pro¹⁷¹ to Gly¹⁷⁸ of SEQ ID NO: 2), in the catalyticdomain, and the 75-amino acid insertion (IS-3: Asp⁵³³ to Val⁶⁰⁷ of SEQID NO: 2) containing the continuous transmembrane-like 24-hydrophobicamino acid sequence, AIAIVIPCILALCLLVLVYTVFQF (SEQ ID NO: 13). Suchthree insertion sequences are present only in MT-MMP-1 among the MMPfamily members but not recognized in the other MMPs. With regard tothree insertion sequences in MT-MMP-3, the number and position ofconstituting amino acid residues thereof is almost same as in MT-MMP-1;however, the amino acid composition thereof is clearly different fromthat of MT-MMP-1, and IS-3 has 37% homology to that in MT-MMP-1.Incidentally, the homology of entire sequences is 53%. A similarsequence to the first insertion IS-1 is exceptionally present in MMP-11;however, the RXKR (SEQ ID NO: 14) sequence conserved in IS-1 is apotential processing region for subtilisin-like enzymes, and it is knownthat the amino acid sequence RXKR (SEQ ID NO: 14) is the subtilisin-likeprotease-cleavage site of various eucaryotic secretory proteins (J.Biol. Chem., 266: 12127 to 12130, 1991). The continuous sequencecomposed of hydrophobic amino acids in IS-3 is believed to be atransmembrane (TM) domain (TM is specifically characteristic of MT-MMP-1(J. Biol. Chem.: 270, 801 to 805, 1995)). Thus, the continuous sequenceof hydrophobic amino acids existing in IS-3 of MT-MMP-3 is also expectedto be a transmembrane domain (see: Example 5). The amino acid sequenceencoded by MT-MMP-3cDNA isolated according to the present invention ishighly homologous to the other MMP family and is similar to MT-MMP-1previously discovered by the present inventors; however, it is obviouslydifferent in the detailed points and differed in molecule weight. Theprotein of the present invention has a molecule weight of approximately69 kDa.

These features on the sequences suggest that MT-MMP-1 and MT-MMP-3 forma sub-family in the MMP family.

Example 2 Expression of MT-MMP-3 mRNA

(a) Expression in Human Tissues

Northern blotting was carried out by using the membrane, Human MultipleTissue Northern Blots (Clontech) onto which poly (A)⁺RNA samples derivedfrom human tissues such as heart, brain, placenta, lung, liver, skeletalmuscle, kidney, and pancreas were applied and by using as a probe the³²P-labeled 2.1 kb cDNA as described in Example 1 (e). Labeling of theprobe was done in the same manner as in Example 1 (c). Multiple TissueNorthern Blots filters wetted with 3× SSC (0.45M NaCl, 0.045M trisodiumcitrate 2H₂O, pH7.0) were dipped in 10 ml of pre-hybridization solution(0.75M NaCl, 2.5 mM EDTA, 0.5× Denhardt's solution, 50% formamide, and20 mM Tris-HCl buffer (pH 7.5) containing 1% SDS). Pre-hybridization wascarried out at 42° C. for two or three hours with gentle stirring. Next,the pre-hybridization solution was exchanged with a solution obtained byaddition of heat-denatured probes to 10 ml of hybridization solution (inwhich 10% sodium dextran and 20 μg/ml denatured salmon sperm DNA wereadded to a pre-hybridization solution). Hybridization was carried out at43° C. overnight. After completion of hybridization, the filters werewashed with a 2× SSC solution containing 0.1% SDS.

Next, the blots were placed in a 1× SSC solution containing 0.1% SDS at55° C. for 30 min. The blots were traced with a Bioimage AnalyzerBAS1000 (Fuji Photo Film Co., Ltd.), and expression intensities of mRNAsin each tissue was assessed. At this time, the same blots were probedwith ³²P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene(CLONTECH) for using as a mRNA internal standard.

The results are shown in FIG. 2A. The size of MT-MMP-3 mRNA is 12 kb inany tissue. Among the tissues examined, a band specific MT-MMP-3 cDNAprobe was detected in lung, brain, and placenta, with high expression;however, it was undetectable in the heart, kidney, liver, pancreas, andskeletal muscle.

On the other hand, when northern blotting was carried out by using HumanMultiple Tissue Northern Blots (Clontech), and using as a probe³²P-labeled MT-MMP-1 cDNA, MT-MMP-1 mRNA detected at 4.5 kb wassignificantly expressed in lung, kidney, and placenta. The lowestexpression occurred in the brain. Cross-hybridization of MT-MMP-1 andMT-MMP-3 was not generated.

(b) Expression in Cultured Tumor Cells

The expression of MT-MMP-3 mRNA in various cultured human tumor celllines was examined. The human tumor cell lines used were larynxcarcinoma-derived Hep2 cell, bladder carcinoma-derived T24 cell, lungcarcinoma-derived PC-3 cell, stomach tumor-derived cells KKLS, NKPS, andMKN-28, osteosarcoma-derived cells SK-ES-1 and U-20S, squamous cellcarcinoma-derived OSC-19, and malignant melanoma A375 cell. Thefibroblasts used were human embryonal lung-derived fibroblasts HEL.

RNA samples extracted from each cells (10 μg per sample) were dissolvedin 2% MOPS (pH 7.5) containing 50% formamide and 17.5% formalin andreacted at 65° C. for 10 min. The products were applied to 1% agarosegel electrophoresis in 2% MOPS. After electrophoresis, the gel wastransferred onto a nylon membrane (for example, Hybond-N, Amersham).After the transfer, the membrane was fixed by irradiating ultravioletrays with 254 nm in wavelength by 1200 micro Joule. The blots werehybridized with ³² P-labeled cDNA for 16 hours in the same manner as inthe foregoing (a), and were traced with a Bioimage Analyzer BAS1000(Fuji Photo Film Co., Ltd.). Signals were detected and their intensitywas assessed.

MT-MMP-3 mRNA was detected in bladder carcinoma T24 and larynx carcinomaHep2 cells with higher expression than in the other cells. However, theexpression of MT-MMP-1 mRNA was at low levels in these cells.

On the other hand, in OSC-19 cells and HEL cells in which thesignificant expression of MT-MMP-1 mRNA was detected, the expressionlevel of MT-MMP-3 mRNA was lower than in other cells (FIG. 2B).

Although MT-MMP-1 and MT-MMP-3 have, from the comparison of the aminoacid sequences, a quite similar domain structure and have the sameaction on activating pro MMP-2 (see: Example 6), expression of the genesfor MT-MMP-1 and MT-MMP-3 shows a completely different pattern in thetissues or cell level. This shows that MT-MMP-1 and MT-MMP-3 are subjectto different expression controls although they have the similarstructure and function.

Example 3 Preparation of Monoclonal Antibodies

(a) Preparation of Antigen Polypeptides

For sequences specific to MT-MMP-3 in which their homology to the otherMMP family is low, the following four sequences were selected from theamino acid sequence of MT-MMP-3 as described in SEQ ID NO: 2 in theSequence Listing, and synthesized:

(SEQ ID NO: 5)

QTRGSSKFHIRRKR

(corresponding to Sequence: Gln¹⁰⁶ to Arg¹¹⁹ of SEQ ID NO: 2;abbreviated as “polypeptide A”)

(SEQ ID NO: 6)

EEVPYSELENGKRD

(corresponding to Sequence: Glu¹⁶⁸ to Asp¹⁸¹ of SEQ ID NO: 2;abbreviated as “polypeptide B”)

(SEQ ID NO: 7)

PTSPRMSVVRSAETMQSA

(corresponding to Sequence: Pro⁵⁵ to Ala⁷² of SEQ ID NO: 2; abbreviatedas “polypeptide C”)

(SEQ ID NO: 8)

TLGNPNHDGNDLFL

(corresponding to Sequence: Thr²²⁹ to Leu²⁴² of SEQ ID NO: 2;abbreviated as “polypeptide D”).

These peptides were synthesized using a peptide synthesizer (peptidesynthesizer 9600, MilliGen/Bioserch) with Fmoc-bop techniques. Cysteinewas introduced on the N-terminus of each polypeptide. The syntheticpeptides were purified with high performance liquid chromatography usingμ Bondasphere, C18 column (Waters).

(b) Preparation of Polypeptide-BSA Conjugates

Each peptide was coupled with bovine serum albumin (BSA) via a cysteineresidue to form an antigen-conjugate. BSA (20 mg) was dissolved in 2 mlof 0.1M phosphate buffer, pH 7.5. Also, 18.13 mg ofN-(6-maleimidocaproyloxy)-succinimide was dissolved in 200 μl ofdimethylformamide. A mixture of the BSA solution and theN-(6-maleimido-caproyloxy)succinimide solution was reacted at 30° C. for30 min., and then subjected to gel filtration through PD-10 (Pharmacia)equilibrated with a 0.1M phosphate buffer, pH 7.0. Maleimido-coupled BSAfractions were collected and concentrated to 1.5 ml or less. Eachsynthetic polypeptide obtained in the above (a) (molar ratio ofpolypeptide: maleimido-coupled BSA=50: 1) was dissolved in 1 ml of 0.1Mphosphate buffer, pH 7.0, then mixed with the maleimido-coupled BSA thusprepared. The mixture was incubated at 4° C. for 20 hours to form aBSA-polypeptide conjugate.

(c) Preparation of Antibody-Producing Cells

An eight-week old female Balb/c mouse was primarily immunized byadministering intraperitoneally 200 μg of each BSA-polypeptide conjugate(conjugate of BSA with any of four polypeptides A, B, C, and D, preparedin the above (b) step) together with complete Freund's adjuvants.Eighteen days later, 200 μg of each BSA-polypeptide conjugate dissolvedin a 0.1M phosphate buffer, pH7.5, was administered intraperitoneally tothe primarily immunized mouse for additional immunization. Further 32days later, 100 μg of each BSA-polypeptide conjugate was administeredintraperitoneally to the mouse for final immunization in the similarmanner to that during additional immunization. Next three days later,the spleen was taken out, and the spleen cell suspension was prepared.

(d) Cell Fusion

(1) The following materials and methods were used:

RPMI-1640 medium:

To RPMI-1640 (Flow Lab.) were added sodium bicarbonate (24 mM), sodiumpyruvate (1 mM), penicillin G potassium (50 U/ml), amikacin sulfate (100μg/ml), and the mixture was adjusted pH to 7.2 with dry ice, sterilizedand filtered through a 0.2 μm Toyo Membrane Filter.

NS-1 medium:

To the above RPMI-1640 medium was added sterilized and filtered FCS (M.A. Bioproducts) until a concentration of FCS reached 15% (v/v).

PEG 4000 solution:

To RPMI-1640 medium was added polyethylene glycol 4000 (PEG 4000, Merck& Co.) until a concentration of PEG 4000 reached 50% (w/w). Thus, theserum-free solution was prepared.

Cell fusion using 8-azaguanine-resistant myeloma SP2 cells (SP2/0-Agl4)was carried out by slightly modified methods according to Oi, et al.techniques disclosed in “Selected Method in Cellular Immunology pp.351to 372 (ed. B. B. Mishell and S. N. Shiigi), W. H. Freeman and Company(1980)”.

(2) Described below is cell fusion between murine nucleated spleen cellsimmunized with polypeptide A-BSA conjugates and myeloma SP2 cells.

The respective nucleated spleen cells (viable cell rate: 100%) preparedin the foregoing (c) were fused with myeloma cells (viable cell rate:100%) in a ratio of 5:1 according to the following procedure:

The polypeptide A-immunized spleen cell suspension and the myeloma cellswere washed respectively with a RPMI 1640 medium followed byresuspending in the same medium. For fusion, 1.1×10⁹ nucleated spleencells and 2.1×10⁸ myeloma cells were mixed together. The cell suspensionwas pelleted by centrifugation and the supernatant fluid was completelyaspirated off. To the cell pellet was added 7.1 ml of PEG 4000 solution(RPMI 1640 medium containing 50% (w/v) polyethylene glycol 4000)pre-warmed to 37° C. dropwise for 1 min., and stirred for 1 min. toallow the cells to be resuspended and dispersed. Next, after 14.2 ml of37° C. pre-warmed RPMI 1640 medium was added dropwise for 2 min., 49.7ml of the same medium was added dropwise within 2 to 3 min. withstirring to allow the cells to be dispersed. This cell dispersion wascentrifuged, and the supernatant fluid was completely aspirated off. Tothe cell pellet was added 71 ml of 37° C. pre-warmed NS-1 medium (RPMI1640 medium supplemented with filtered sterile 15% (w/v) fetal calfserum (JRH Bioscience)) quickly, and a large cell mass was carefullydispersed by pipetting. Next, the cell suspension was diluted with 142ml of the same medium, and 6.0×10⁵ cells/0.1 ml was plated on each wellof a polystyrene 96-well microtiter tray. The cell-containing microwellwas incubated at 37° C. under a 100% humidified atmosphere containing 7%CO₂/93% air.

For mouse-derived spleen cells immunized with the polypeptide B-BSAconjugate, the spleen cells (6.2×10⁸ cells) were mixed with 1.24×10⁸myeloma cells, and PEG 4000 solution, RPMI 1640 medium, and NS-1 mediumas used above were used by 4.1 ml, 36.9 ml, and 123 ml, respectively.

For mouse-derived spleen cells immunized with the polypeptide C-BSAconjugate, the spleen cells (3.6×10⁸ cells) were mixed with 7.5×10⁷myeloma cells, and PEG 4000 solution, RPMI 1640 medium, and NS-1 mediumwere used by 2.5 ml, 22.5 ml, and 75 ml, respectively.

For mouse-derived spleen cells immunized with the polypeptide D-BSAconjugate, the spleen cells (6.0×10⁸ cells) were mixed with 1.2×10⁸myeloma cells, and PEG 4000 solution, RPMI 1640 medium, and NS-1 mediumwere used by 4.0 ml, 36.0 ml, and 120 ml, respectively.

(e) Selective Growth of Hybridomas in Selection Medium

(1) Media to be used were as follows:

HAT medium: To NS-1 medium as described in foregoing

(d) (1) was added further hypoxanthine (100 μM), aminopterin (0.4 μM),and thymidine (16 μM).

HT medium: The medium has the same composition as the foregoing HATmedium except that aminopterin was excluded.

(2) Next day (first day) from culture initiation of the foregoing (d),two drops of HAT medium (approximately 0.1 ml) was added to the cellswith a Pasteur pipette. On the 2nd, 3rd, 5th, and 8th days, a half ofthe medium (approximately 0.1 ml) was replaced with fresh HAT medium,respectively. On the 11th day, a half of the medium was replaced withfresh HT medium. On the 14th day, positive wells were examined by solidphase-antibody binding test (enzyme-linked immunosorbent assay; ELISA)for all wells wherein the growth of hybridomas was visually recognized.

Polystyrene 96-well plates were coated with polypeptides A, B, C, and D,respectively, used as an antigen, and washed with PBS (containing 0.05%Tween 20) for washing to remove unadsorbed peptides. Next, the uncoatedportions of each well were blocked with 1% BSA. To eachpolypeptide-coated well was added 0.1 ml of supernatant fluid from thehybridoma well in which hybridomas were grown and the polypeptide-coatedwell was allowed to stand at room temperature for approximately onehour. To the polypeptide-coated well was added, as a second antibody,horseradish peroxidase (HRP)-labeled goat anti-mouse immunoglobulin(Cappel Lab.), and the well was further allowed to stand at roomtemperature for approximately 1 hour. Next, to the well was addedsubstrates, hydrogen peroxide and o-phenylenediamine, and OD readings at492 nm were obtained by a microplate OD reader (MRP-A4, Toso, Japan).

(f) Cloning of Hybridomas

Hybridomas in the well positive against each antigen peptide obtained inthe foregoing (e) were cloned by limiting dilution to establishmonoclones.

That is, a cloning medium containing, as feeder cells, 10⁷ mousethymocytes per 1 ml of NS-1 medium was prepared. Into a 96-wellmicrotiter tray was plated hybridomas at a cell density of 5, 1, or 0.5cells per well, respectively, with dilutions wherein the 5, 1, or 0.5hybridoma cells per well was plated to 36, 36, and 24 wells,respectively. On the 5th and 12th days, about 0.1 ml of NS-1 medium wasadded to all the wells. Approximately two weeks later from theinitiation of cloning, ELISA as described in the above (e) was conductedfor groups wherein the sufficient growth of hybridomas was visuallyrecognized and the rate of colony formation-negative wells is 50% ormore. In cases where all the examined wells were negative, 4 to 6 wellseach containing 1 colony were selected from antibody-positive wells, andrecloned. Finally, as shown in Tables 1 to 4, 7 anti-polypeptide Aantibody-producing, 16 anti-polypeptide B antibody-producing, 11anti-polypeptide C antibody-producing, and 4 anti-polypeptide Dantibody-producing hybridoma cells were obtained, respectively.

(g) Cultivation of hybridomas and Purification of Monoclonal Antibodies

Each hybridoma cell thus obtained was cultured in NS-1 medium to affordmonoclonal antibodies with a concentration of 10 to 100 μg/ml in theculture supernatant. Further, 10 hybridoma cells thus obtained wereadministered intraperitoneally to a mouse (inbred BALB/c mouse, ♀,six-week old) intraperitoneally primed with pristane 1 week prior toinjection, and two weeks later an ascitic fluid containing 4 to 7 mg/mlmonoclonal antibody was recollected. The obtained ascitic fluids weresalted out with 40% ammonium sulfate saturation, IgG class antibodieswere adsorbed on protein A affigel (Bio-Rad), followed by elution with a0.1M citrate buffer (pH 5) to afford purified forms.

(h) Determination of Class and Sub-class for Monoclonal Antibody

To microtiter plates on which polypeptides A, B, C, and D were coatedaccording to ELISA as described herein above, was added each supernatantobtained in the above (f). Next, after PBS washing, iso-type specificrabbit anti-mouse IgG antibodies (Zymed Lab.) was added. After PBSwashing, horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) wasadded, and visualization was carried out with hydrogen peroxide and 2,2′-azinodi(3-ethylbenzothiazolinic acid). As a result, the class andsub-class were determined. Finally, as shown in Tables 1 to 4, pluralmonoclonal anti-MT-MMP-3 antibody-producing hybridomas were obtained.

TABLE 1 Polypeptide Clone No. Subclass/Chain A 116-1E7 γ1/κ 116-2G6 γ1/κ116-6A11 γ1/κ 116-7B2 μ/κ 116-10E10 μ/κ 116-11B2 μ/κ 116-12E3 μ/κ

TABLE 2 Polypeptide Clone No. Subclass/Chain B 117-1F6 γ1/ κ 117-2H5 γ1/κ 117-3B9 γ1/ κ 117-4E1 γ1/ κ 117-5A6 γ1/ κ 117-6C11 γ1/ κ 117-9H5 γ1/ κ117-10C6 γ1/ κ 117-13B6 γ2a/ κ 117-14E3 γ1/ κ 117-15C5 γ1/ κ 117-16E10γ1/ κ 117-17E10 γ2b/ κ 117-18D9 γ1/ κ 117-19D1 γ1/ κ 117-20B3 γ1/ κ

TABLE 3 Polypeptide Clone No. Subclass/Chain C 157-3G4 γ1/ κ 157-4A5γ2b/ κ 157-6F5 γ1/ κ 157-11E1 μ/ κ

TABLE 4 Polypeptide Clone No. Subclass/Chain D 158-2D6 γ2a/ κ 158-3E12γ2a/ κ 158-8E6 γ1/ κ 158-9F6 γ2b/ κ 158-11D10 μ/ κ 158-16F12 γ1/ κ158-17F1 γ1/ κ 158-18D8 γ1/ κ 158-19F10 γ1/ κ 158-20D5 γ2a/ κ 158-21F11γ1/ κ

The clone No. 117-4E1 has been deposited as from Jul. 5, 1995 (originaldeposit date) with NIBH and has been assigned the Accession Number FERMP-15031. The original deposit of the hybridoma 117-4E1 has beentransferred to one under the Budapest Treaty by a request dated Jul. 1,1996 and is on deposit with the Accession Number FERM BP-5572 under theterms of the Budapest Treaty at NIBH.

(i) Specificity of Anti-MT-MMP-3 Monoclonal Antibody

The cross-reactivity of each monoclonal anti-MT-MMP-3 antibodies (cloneNos. 117-4E1, 157-6F5 and 158-8E6) wherein each antibody positivelyreacts with a human MT-MMP-3 peptide was examined by solidphase-antibody binding tests (ELISA), as described in the above (e),using as an antigen pro MMP-1 (Clin. Chim. Acta, 219: 1 to 14, 1993),pro MMP-2 (Clin. Chim. Acta, 221: 91 to 103, 1993), and pro MMP-3 (Clin.Chim. Acta, 211: 59 to 72, 1992), purified from the culture supernatantof human embryonal fibroblasts (NB1RGB), respectively; pro MMP-7purified from the culture supernatant of human rectal carcinomas (CaR-1)(Cancer Res., 50: 7758 to 7764, 1990); pro MMP-8 from human neutrophiles(Biol. Chem. Hoppe-Seyler, 371: Supplement, 295 to 304, 1990); or proMMP-9 from the culture supernatant of human fibrosarcomas (HT1080),respectively.

That is, polystyrene 96-well plates were used. Purified MMP-1, MMP-2,MMP-3, MMP-7, MMP-8, and MMP-9 were added in 50 ng/well to each well,respectively, to coat the well. After the wells were washed with PBS forwashing to remove unadsorbed antigens, uncoated portions of each wellwere blocked with PBS containing 3% skim milk. To each well was addedeach anti-MT-MMP monoclonal antibody with 1 μg/well, and the well wasallowed to stand at room temperature for approximately 1 hour. After theplates were washed, peroxidase-labeled goat anti-mouse immunoglobulinwas added as a second antibody and further was reacted at roomtemperature for approximately 1 hour. Next, substrates, hydrogenperoxide and o-phenylenediamine, were added, and optical density (OD)readings at 492 nm were obtained by a microplate OD reader (MRP-A4,Toso, Japan).

As a result, none of the anti-MT-MMP-3 monoclonal antibodies hadreactivity with purified MMPs samples, other than MT-MMP-3.

The methods as described in Example 3 are repeated, by using, as anantigen, recombinant MT-MMP-3 , for example, recombinant MT-MMP-3obtained in Examples 4 and 5 described herein below, instead of thesynthetic peptide antigen, to produce monoclonal anti-MT-MMP-3antibodies similarly.

Example 4 Expression and Identification of Gene Products

To express MT-MMP-3 in animal cells as hosts, cDNA was ligated with anexpression vector.

In this Example, pSG5 (Stratagene) containing the SV40 promoter,enhancer, poly A signal, small T antigen gene intervening sequence wasused for the expression vector. The recombinant pbluescript™(Stratagene) wherein cloned MT-MMP-3 gene was integrated and which wereconstructed in Example 1 (e) was cleaved with EcoRI to produce 2.1 kbinsertion fragments which were inserted into the EcoRI site ofeukaryotic expression vector pSG5 to form expression plasmid pSGMT2.Ligation was carried out in accordance with the protocols attached withligation kits. African green monkey kidney-derived COS-1 cells werecultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with5% fetal calf serum and 2 mM glutamine. The cultured COS-1 cells werecotransfected with pSGMT2 and pSGT1 (TIMP-1 cDNA was cloned in pSG5)according to calcium phosphate techniques (Virology, 52: 456, 1973). Fora control, COS-1 was transfected with pSG5 alone.

That is, to distilled water was added 2 μg of recombinant pSG5 or pSG5alone, to which 60 μl of 0.25M CaCl₂ was added. Then 62.5 μl of 2× BBSsolution (50 mM BES buffer (pH7.9) containing 2.8 mM Na₂HPO₄ and 280 mMNaCl) was added to the bottom of the tube. After mixing, the mixture wasallowed to stand at room temperature for 30 minutes, until sufficientprecipitation occurred. After the precipitates were dispersed bypipetting and added dropwise to COS-1 cells, the resultant cells wereincubated in a CO₂ incubator for approximately 24 hours. Then the mediumwas removed, the cells were washed with PBS, followed by addition offresh methionine-free DMEM supplemented with 30 μCi/ml 5S-methionine.The cultivation was continued for 5 hours to label cell proteins with³⁵S. The cells and condition medium were separated by centrifugation,and the cells were incubated at 4° C. for 1 hour in a lysis buffersolution (10 mM Tris-HCl buffer, pH7.5, containing 0.15M NaCl, 0.1%sodium deoxycholate, 0.1% SDS, 1 mM Triton X-100, 1% NP-40, 1 mM EDTA,lmM phenylmethanesulfonyl fluoride (PMSF)). The cell lysates werecentrifuged to recollect supernatants. Both the cell lysate supernatantsand conditioned medium were reacted with anti-MT-MMP-3 polypeptideantibody clones Nos. 117-4E1 or 117-13B6 (obtained in Example 3), andfor a control with anti-TIMP-1 antibody clone No. 50-1H7 at 4° C. for 16hours. Clone Nos. 117-4E1 or 117-13B6 antibody were selected becausethey have low non-specific reactivity among the anti-MT-MMP-3 monoclonalantibodies. To these antigen-antibody complexes was added proteinA-coupled Sepharose™-4B (Pharmacia) and the mixture was incubated at 4°C. for 2 hours with stirring to carry out immunoprecipitation. Then, theSepharose™-4B coupled with immunoprecipitated monoclonal antibodies wasprecipitated by centrifugation, and washed three times with a lysisbuffer solution, and finally with a 0.05M Tris-HCl buffer solution,pH6.8. To this washed Sepharose™-4B was added a SDS polyacrylamideelectrophoresis sample buffer solution (50 mM Tris-HCl buffer (pH 6.5)containing 10% glycerol, 2% SDS, 2% β-mercaptoethanol, 0.1% bromophenolblue) and the mixture was heated at 100° C. for 3 min., and then appliedto 12% SDS polyacrylamide electrophoresis. After electrophoresis, thegel was detected using a Bioimage Analyzer BAS1000 (Fuji Photo Film Co,.Ltd.). The results are shown in FIG. 3.

Both anti-MT-MMP-3 polypeptide mAbs 117-4E1 and 117-13B6 used wereprecipitated immunologically a 64-kDa protein specifically from thelysate of cells transfected with MT-MMP-3 genes. Neither of the mAbs wasprecipitated from that of cells transfected the control vector pSGSwherein no MT-MMP-3 gene was included. The molecular size 64 kDa of theproteins detected in immunoprecipitation almost matched the moleculeweight calculated from the amino acid sequence of SEQ ID NO: 2 inSequence Listing.

In addition, three bands equivalent to molecular sizes 30, 33, and 52kDa were detected only from cell lysates of cells transfected withMT-MMP-3 genes. However, none of these bands were detected in thecontrol.

On the other hand, none of these proteins as immunoprecipitated fromcell lysates were detected from the conditioned culture medium. To thecontrary, TIMP-1 was a secretory protein. In fact, most of the expressedTIMP-1 was detected in the conditioned culture medium and it wasconfirmed that TIMP-1 was surely secreted outside the cells.

The foregoing results show that MT-MMP-3 is not easily secreted thoughthe presence of a signal peptide is suggested from its amino acidsequence. This finding is very similar to the previous finding obtainedby the present inventors in which MT-MMP-1 was expressed on the cellsurface layer, but was not detected in the culture medium (Nature, 370;61 to 65, 1994).

Since MT-MMP-3 cDNA is a full-length cDNA synthesized withreverse-transcriptase from mRNA, MT-MMP-3 can be mass-produced viatransferring this cDNA to a suitable expression vector wherein E. coli,bacillus subtilis, yeasts, animal cells or the like are used as a host.In the Example in which pSGMT2 was introduced into COS-1, MT-MMP-3 istransiently expressed in the transformant COS-1; however, cell strainscapable of producing the targets for a long period can be obtained usingexpression vectors having a suitable selection marker (for example, neogenes, dehydrofolate reductase genes, etc.) and introducing it into CHOcells or the like.

Example 5 Function of the C-terminal Hydrophobic Amino Acid ContinuousSequence of MT-MMP-3

(a) Preparation of Chimeric Protein (TIMP/TM-3) between MT-MMP-3C-Terminal Hydrophobic Amino Acid Continuous Sequence and TIMP-1 and ofChimeric Protein (TIMP/MT-1) between MT-MMP-1 C-Terminal HydrophobicAmino Acid Continuous Sequence and TIMP-1

Preparation of chimeric proteins between MT-MMP C-terminal hydrophobicamino acid continuous sequence and TIMP-1 was carried out according totechniques for preparation of chimeric proteins between MT-MMP-1transmembrane domain and TIMP-1 in Cao, et al. (J. Biol. Chem. 13; 801to 805, 1995).

cDNA fragments encoding the the amino acid sequence (Ala⁵⁵⁹ to Val⁶⁰⁷)containing MT-MMP-3 C-terminal hydrophobic amino acid sequence wereamplified by PCR techniques and recollected. Similarly, cDNA fragmentsencoding the the amino acid sequence (Gly⁵³⁵ to Val⁵⁸² ) containingMT-MMP-1 C-terminal hydrophobic amino acid continuous sequence wereamplified by PCR and recollected. PCR amplification was carried out inthe similar manner to that in Example 1(b).

Each DNA fragment thus obtained was ligated into the 3′-terminal side ofTIMP-1 cDNA, and subjected to subcloning to pSG5. Thus, expressionplasmid pSGTIM2 for TIMP-1/MT-3 chimeric protein was produced.Similarly, expression plasmid pSGTIM1 for TIMP-l/MT-1 chimeric proteinwas produced. Ligation was carried out in accordance with the protocolsaccompanying with the ligation kit.

Transfection of these plasmids into COS-1 was carried out in the similarmanner to that described in Example 4. COS-1 cells cultured in DMEMsupplemented with 5% fetal calf serum and 2 mM glutamine weretransfected with PSGT1M2, pSGT1M1, and pSGT1, respectively, by thecalcium phosphate technique. As a control, COS-1 was transfected withpSG5 alone.

That is, to 2 μg of plasmid DNA was added 60 μl of 0.25M CaCl₂. Then62.5 μl of 2× BBS solution (50 mM BES buffer, pH7.9 containing 2.8 mMNa₂HPO₄ and 280 mM NaCl) into the bottom of the tube. After mixing, themixture was allowed to stand at room temperature for approximately 30min to form precipitates sufficiently. The precipitates were dispersedby pipetting and added dropwise to COS-1 cells, and then the mixture wasincubated in a CO₂ incubator for approximately 24 hours. After removalof the medium, the cells were washed with PBS, to which then freshmethionine-free DMEM containing ³⁵S-methionine was added. Thecultivation was continued for 5 hours to label cell proteins with ³⁵S.

The cells and the conditioned culture medium were separated from eachother by centrifugation, and the cells were incubated at 4° C. for 1hour in a lysis buffer solution (10 mM Tris-HCl buffer, pH 7.5containing 0.15M NaCl, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM TritonX-100, 1% NP-40, 1 mM EDTA, and 1 mM PMSF), and supernatants werecollected. The lysed cell supernatants and the conditioned culturemedium were reacted with anti-TIMP-1 antibody, clone No. 50-1H7(obtained in Example 3), at 4° C. for 16 hours.

To the antigen-antibody complexes thus obtained was added a proteinA-coupled Sepharose™-4B (Pharmacia) and the mixture was incubated withstirring at 4° C. for 2 hours to carry out immunoprecipitation. Theimmunoprecipitated Sepharose™-4B coupled with the monoclonal antibodywas precipitated by centrifugation, the precipitate was washed 3 timeswith a lysis solution, and then finally washed with 0.05M Tris-HClbuffer, pH 6.8. To this washed Sepharose™-4B was added an SDSpolyacrylamide electrophoresis sample buffer solution (50 mM Tris-HClbuffer, pH 6.5 containing 10% glycerol, 2% SDS, 2% β-mercaptoethanol,0.1% bromophenol blue) and the mixture was heated at 100° C. for 3 min.,and then applied to 12% SDS polyacrylamide gel electrophoresis. Afterthe electrophoresis, signals of the gel were detected by a BioimageAnalyzer-BAS1000 (Fuji Photo Film Co., Ltd.).

TIMP-1, TIMP-1/MT-1, and TIMP-1/MT-3 were detected as 28, 32, and 32 kDaproteins in the cell lysate, respectively. The molecule sizes of thedetected chimeric proteins TIMP-1/MT-1 and TIMP-1/MT-3 matched themolecule weights estimated from the construct of the fused genes. TIMP-1was predominantly detected in the conditioned culture medium, though itwas found in the cell lysate. However, TIMP-1/MT-1 was detectedexclusively in the cell lysate, but not in the conditioned culturemedium (J. Biol. Chem., 13; 801 to 805, 1995). TIMP-1/MT-3 was detectedexclusively from the cell lysate, similar to TIMP-1/MT-1. Thelocalization of TIMP-1/MT-3 was exactly the same as that of TIMP-1/MT-1(FIG. 4).

These results show that the hydrophobic amino acid continuous sequenceat the MT-MMP-3 C-terminal region suppresses secretion of the fusionproteins to the outside of the cells with a function similar to thehydrophobic amino acid continuous sequence at the MT-MMP-1 C-terminalregion.

(b) Expression of Chimeric Proteins in Cell Surface Layers

It was examined whether in fact the hydrophobic amino acid continuoussequence in the MT-MMP-3 C-terminal region is functioning as atransmembrane domain by indirect immunofluorescence staining forTIMP-1/MT-3 expression cells. COS-1 cells were transfected with pSGT1 orpSGT1M2 by the calcium phosphate technique in the similar manner to thatdescribed in Example 4. In this Example, the cells were cultured on aslide chamber without using an isotope-labeled medium. After 24-hourculturing, the cells were reacted at 37° C. for 40 minutes in PBScontaining 5 μg/ml anti-TIMP-1 antibody, clone No. 50-1H7, and 3% BSA.Then the cells were washed three times with PBS containing 3% BSA,air-dried, and fixed with acetone for 5 min. The cells were soaked inPBS containing 3% BSA, and then reacted with 1500× diluted fluoresceinisothiocyanate (FITC)-conjugated goat anti-(mouse IgG) IgG (Capel) at37° C. for 30 min. Then an excessive amounts of antibodies was washedout with PBS containing 3% BSA. Finally, the specimens were overlaidwith glycerin and observed under a immunofluorescence microscope.

As a result, in pSGTlM2-expressing cells (chimeric proteinTIMP-1/MT-3-producing cells), fluorescence was observed on the cellsurface, confirming that the TIMP-1 portion of the chimeric protein wasexpressed on the cell surface layer. On the other hand, no fluorescencewas observed in pSGT1-expressing cells (non-chimeric TIMP-1-producingcells), and the expression of TIMP-1 was not observed on the cellsurface layer (FIG. 5).

This result shows that the MT-MMP-3 C-terminal hydrophobic amino acidcontinuous sequence is functioning as a transmembrane (TM) domain.

Example 6 Activation of Pro MMP-2 due to Expression of MT-MMP-3

COS-1 cells were cotransfected with plasmid pSG5M2 for MT-MMP-3 cDNA asconstructed in Example 4, plasmid pSG5M1 for MT-MMP-1 cDNA, or vectorpSG5, respectively, together with plasmid PSGGA for pro MMP-2, by thecalcium phosphate technique as described in Example 4. In theexperiments, a conventional fresh medium was used instead of a freshmedium containing ³⁵S-methionine. In addition, human fibrosarcomaHT-1080 cell lines were cotransfected with pSGT1, pSGT2, or pSG5,respectively, together with pSGM2. In the immunoprecipitationexperiments, it was confirmed that transformed HT-1080 cell lines secretpro MMP-2 and pro MMP-9 constitutively (corresponding to 68 kDa and 97.4kDa bands, respectively, in FIG. 6), and MT-MMP-3 cDNA-transfected cellsexpress MT-MMP-3 (see: Example 4).

The transfectants thus obtained were cultured for 24 hours in serum-freeDMEM, and the recollected culture supernatants were applied tozymography. The culture supernatants were mixed with a SDSpolyacrylamide electrophoresis sample buffer solution (no-reducingagent; 50 mM Tris-HCl buffer, pH 6.5 containing 10% glycerol, 2% SDS,0.1% bromophenol blue) and the mixture was incubated at 37° C. for 20min., and applied to electrophoresis employing the following conditions:20 mA, 4° C., 10% polyacrylamide gel containing 0.1% gelatin.

After electrophoresis, the gel was washed in a 2.5% Triton X-100solution with gentle shaking for 1 hour, and then incubated in agelatinase buffer solution (50 mM Tris-HCl, pH7.6 containing 10 mMCaCl₂, 0.15M NaCl, and 0.02% NaN₃ with slow shaking at 37° C. for 24hours. The buffer solution was discarded, and the gel was stained in0.1% Coomassie Brilliant Blue R250 (dissolved in 50% methanol-10% aceticacid) for 1 hour, then was soaked in a decoloring solution (5% methanol-7.5% acetic acid) and decolored. The results of the zymography was shownin FIG. 6.

Similarly to that in MT-MMP-1 cDNA-transfected COS-1, 64 kDa and 62 kDabands corresponding to activate intermediate MMP-2 and active MMP-2,respectively, were newly expressed in MT-MMP-3 cDNA-transfected COS-1.Thus, the activation of pro MMP-2 was confirmed. On the other hand, invector pSG5-transfected cells, only 68 kDa band of pro MMP-2 wasdetected, but the molecule size change accompanied with activation wasnot observed (FIG. 6A).

For COS-1 cells, the pro MMP-2 activation due to the expression plasmidwas observed by cotransfection of pro MMP-2 expression plasmid (pSGGA).For HT1080 that constitutively expresses pro MMP-2, the pro MMP-2activation accompanied with MT-MMP-3 expression was observed. The activeform pro MMP-2 observed in this HT1080 has the same molecular size asthe pro MMP-2 molecule induced by treating HT1080 cells with 100 μg/mlconcanavalin A, and was specifically reacted with monoclonal anti-MMP-2antibody. This activation was not observed in control cells transfectedwith the vector alone. On the other hand, for pro MMP-9, the change ofthe molecule size was not recognized as was in the control cells, andthe activation was not recognized.

The activation of pro MMP-2 was suppressed in TIMP-1 andMT-MMP-3-cotransfected cells. It was suppressed in TIMP-2 andMT-MMP-3-cotransfected cells, too. The inhibitory degree in the TIMP-2co-transfected cells was more significant than that in the TIMP-1, andthis tendency was similar in MT-MMP-1 and MT-MMP-3 (FIG. 6B).

In an embodiment, the present invention relates to:

(A) a protein or a salt thereof, which (i) has an activity identicalwith or substantially equivalent to native MT-MMP, (ii) is a member ofMMPs having the capability of activating pro MMP-2, (iii) is anactivator for pro MMP-2, and (iv) is different from MT-MMP-1;

(B) the protein according to the above (A), wherein the protein has anactivity or a primary structural conformation identical with orsubstantially equivalent to that of MT-MMP-3 or a salt thereof;

wherein the protein has activity substantially equivalent to MT-3MMP-3or its salt or has the primary structure conformation substantiallyequivalent thereto;

(C) the protein according to the above (A) or (B), wherein a C-terminalarea of the protein has (i) an amino acid sequence from Ala⁵⁶⁴ to Phe⁵⁸⁷in the sequence represented by SEQ ID NO: 2 in the Sequence Listing or(ii) an amino acid sequence substantially equivalent thereto;

(D) the protein according to any of the above (A) to (C), wherein theprotein is MT-MMP-3 or a salt thereof which has (i) an amino acidsequence represented by SEQ ID NO: 2 in the Sequence Listing or (ii) anamino acid sequence equivalent thereto;

(E) the protein according to any of the above (A) to (D), wherein theprotein is a product obtained by expressing a foreign DNA sequence inprokaryotes or eukaryotes;

(F) the protein according to any of the above (A) to (E), wherein theprotein has (i) the amino acid sequence of SEQ ID NO: 2 in the SequenceListing or (ii) the substantially same amino acid sequence;

(G) a partial peptide (or a peptide fragment) or its salt of the proteinaccording to any of the above (A) to (F);

(H) a nucleic acid comprising a nucleotide sequence coding for theprotein according to any of the above (A) to (F) or a partial peptidethereof;

(I) the nucleic acid according to the above (H) which is a DNA genehaving a nucleotide sequence coding for MT-MMP-3 according to any of theabove (B) to (D);

(J) the nucleic acid according to the above (H) or (I), having (i) anopen reading frame region of the nucleotide sequence represented by SEQID NO: 1 in the Sequence Listing or (ii) a nucleotide sequence having anactivity substantially equivalent thereto;

(K) a vector comprising the nucleic acid according to any of the above(H) to (J);

(L) a transformant wherein the nucleic acid according to any of theabove (H) to (J) or the vector according to the above (K) is harbored;and

(M) a process for producing the protein or its partial peptide accordingto any of the above (A) to (F), which comprises:

(i) culturing the transformant according to the above (L) in a nutrientmedium capable of growing said transformant, and

(ii) producing, as a recombinant species, the protein or its partialpeptide according to any of the above (A) to (F), including MT-MMP-3 ora salt thereof.

Such a protein or a partial peptide thereof, and a nucleic acid arelabeled and can be used for measurement and examination.

In another embodiment, the present invention relates to:

(a) a method for producing an antibody against a species selected fromthe group consisting of a protein or a salt thereof and a peptidethereof or a salt thereof according to any of Claims 1 to 6, includingMT-MMP-3 or a salt thereof,

which comprises employing an antigen selected from the group consistingof said protein, said partial peptide and a salt thereof, and MT-MMP-3or a salt thereof to raise the antibody thereagainst;

(b) an antibody against a species selected from the group consisting ofa protein or a salt thereof according to any of claims 1 to 6, andMT-MMP-3 or a salt thereof,

(c) the antibody according to the above (b), wherein the antibody is ananti-serum;

(d) the antibody according to the above (b), wherein the antibody ismonoclonal;

(e) the antibody according to the above (b) or

(d), which is a monoclonal antibody against MT-MMP-3 or a salt thereof;

(f) a method for producing the antibody according to above (d) or (e),which comprises

(1) fusing an antibody-producing cell obtained from an immunized animalwith an immortal cell, wherein said animal is immunized with a speciesselected from the group consisting of a protein or a salt thereofaccording to any of Claims 1 to 6, a partial peptide of said protein ora salt thereof, and MT-MMP-3 or a salt thereof, and

(2) selecting an immortal hybrid cell capable of an antibody against aspecies selected from the group consisting of a protein or a saltthereof according to any of Claims 1 to 6, and MT-MMP-3 or a saltthereof;

(g) a method for detecting and/or measuring MT-MMP-3, which comprisesusing (A) a reagent selected from the group consisting of a protein or asalt thereof according to any of claims 1 to 6 and a partial peptide ofsaid protein or a salt thereof, or (B) a reagent selected from the groupconsisting of the antibodies according to any of above (b) to (e);

(h) a labeled antibody against MT-MMP-3 for the method for detectingand/or measuring MT-MMP-3 (the detection and/or measurement of MT-MMP-3)according to above (g);

(i) a labeled protein or a salt thereof, for the method for detectingand/or measuring MT-MMP-3 (the detection and/or measurement of MT-MMP-3)according to above (g), wherein said labeled protein is a speciesselected from the group consisting of a protein or a salt thereofaccording to any of Claims 1 to 6, and MT-MMP-3 or a salt thereof, or alabeled partial peptide of said protein or a salt thereof, for themethod according to above (g);

(j) a labeled nucleic acid for detection andlor measurement of MT-MMP-3expressing cells and/or tissues, wherein said nucleic acid is a speciesaccording to any of claims 8 to 10; and

(k) the nucleic acid according to above (j), which is a probe forhybridization.

The protein or a salt thereof which (i) has an activity identical withor substantially equivalent to native MT-MMP that is a member of MMPcapable of activating pro MMP-2, excluding MT-MMP-1, and (ii) is anactivator for pro MMP-2 can be provided. Further, the nucleic acidencoding such proteins can be obtained. As a result, the diagnosticmeans useful for research & development regarding diagnosis and therapyof cancers including diagnosis of the presence or absence of tumorcells, estimation of malignancy of cancers or the like are provided.These are useful for the other medical and physiological applications.According to the present invention, there is provided: a novel matrixmetalloproteinase specifically expressed on the in cell surface layer ofhuman tumors in particular; a DNA containing a nucleotide sequencecoding for said matrix metalloproteinase; host cells transformed withsaid DNA; a process for producing the matrix metalloproteinase using thetransformed host cells; a monoclonal antibody specifically binding withthe matrix protease protein; and use of these proteins and antibodies.These enable us to investigate a matrix protease specifically expressedon the cell surface layer as a target of anti-metastatic drugs and as amarker for detection of cancers, judgment of malignancy, diagnosis ofcancers, etc. In addition, the present invention is helpful for researchof Alzheimer's diseases. Effective detection and therapeutic means isprovided according to the present invention.

14 2116 Nucleic acid Double Linear cDNA Human 1 GGCTCCTTAC CCACCCGGAGACTTTTTTTT GAAAGGAAAC TAGGGAGGGAGGGAGAGGGA 60 GAGAGGGAGA AAACGAAGGGGAGCTCGTCC ATCCATTGAA GCACAGTTCA CT ATG 115 Met 1 ATC TTA CTC ACA TTCAGC ACT GGA AGA CGG TTG GAT TTC GTG CAT CAT 163 Ile Leu Leu Thr Phe SerThr Gly Arg Arg Leu Asp Phe Val His His 5 10 15 TCG GGG GTG TTT TTC TTGCAA ACC TTG CTT TGG ATT TTA TGT GCT ACA 211 Ser Gly Val Phe Phe Leu GlnThr Leu Leu Trp Ile Leu Cys Ala Thr 20 25 30 GTC TGC GGA ACG GAG CAG TATTTC AAT GTG GAG GTT TGG TTA CAA AAG 259 Val Cys Gly Thr Glu Gln Tyr PheAsn Val Glu Val Trp Leu Gln Lys 35 40 45 TAC GGC TAC CTT CCA CCG ACT GACCCC AGA ATG TCA GTG CTG CGC TCT 307 Tyr Gly Tyr Leu Pro Pro Thr Asp ProArg Met Ser Val Leu Arg Ser 50 55 60 65 GCA GAG ACC ATG CAG TCT GCC CTAGCT GCC ATG CAG CAG TTC TAT GGC 355 Ala Glu Thr Met Gln Ser Ala Leu AlaAla Met Gln Gln Phe Tyr Gly 70 75 80 ATT AAC ATG ACA GGA AAA GTG GAC AGAAAC ACA ATT GAC TGG ATG AAG 403 Ile Asn Met Thr Gly Lys Val Asp Arg AsnThr Ile Asp Trp Met Lys 85 90 95 AAG CCC CGA TGC GGT GTA CCT GAC CAG ACAAGA GGT AGC TCC AAA TTT 451 Lys Pro Arg Cys Gly Val Pro Asp Gln Thr ArgGly Ser Ser Lys Phe 100 105 110 CAT ATT CGT CGA AAG CGA TAT GCA TTG ACAGGA CAG AAA TGG CAG CAC 499 His Ile Arg Arg Lys Arg Tyr Ala Leu Thr GlyGln Lys Trp Gln His 115 120 125 AAG CAC ATC ACT TAC AGT ATA AAG AAC GTAACT CCA AAA GTA GGA GAC 547 Lys His Ile Thr Tyr Ser Ile Lys Asn Val ThrPro Lys Val Gly Asp 130 135 140 145 CCT GAG ACT CGT AAA GCT ATT CGC CGTGCC TTT GAT GTG TGG CAG AAT 595 Pro Glu Thr Arg Lys Ala Ile Arg Arg AlaPhe Asp Val Trp Gln Asn 150 155 160 GTA ACT CCT CTG ACA TTT GAA GAA GTTCCC TAC AGT GAA TTA GAA AAT 643 Val Thr Pro Leu Thr Phe Glu Glu Val ProTyr Ser Glu Leu Glu Asn 165 170 175 GGC AAA CGT GAT GTG GAT ATA ACC ATTATT TTT GCA TCT GGT TTC CAT 691 Gly Lys Arg Asp Val Asp Ile Thr Ile IlePhe Ala Ser Gly Phe His 180 185 190 GGG GAC AGC TCT CCC TTT GAT GGA GAGGGA GGA TTT TTG GCA CAT GCC 739 Gly Asp Ser Ser Pro Phe Asp Gly Glu GlyGly Phe Leu Ala His Ala 195 200 205 TAC TTC CCT GGA CCA GGA ATT GGA GGAGAT ACC CAT TTT GAC TCA GAT 787 Tyr Phe Pro Gly Pro Gly Ile Gly Gly AspThr His Phe Asp Ser Asp 210 215 220 225 GAG CCA TGG ACA CTA GGA AAT CCTAAT CAT GAT GGA AAT GAC TTA TTT 835 Glu Pro Trp Thr Leu Gly Asn Pro AsnHis Asp Gly Asn Asp Leu Phe 230 235 240 CTT GTA GCA GTC CAT GAA CTG GGACAT GCT CTG GGA TTG GAG CAT TCC 883 Leu Val Ala Val His Glu Leu Gly HisAla Leu Gly Leu Glu His Ser 245 250 255 AAT GAC CCC ACT GCC ATC ATG GCTCCA TTT TAC CAG TAC ATG GAA ACA 931 Asn Asp Pro Thr Ala Ile Met Ala ProPhe Tyr Gln Tyr Met Glu Thr 260 265 270 GAC AAC TTC AAA CTA CCT AAT GATGAT TTA CAG GGC ATC CAG AAA ATA 979 Asp Asn Phe Lys Leu Pro Asn Asp AspLeu Gln Gly Ile Gln Lys Ile 275 280 285 TAT GGT CCA CCT GAC AAG ATT CCTCCA CCT ACA AGA CCT CTA CCG ACA 1027 Tyr Gly Pro Pro Asp Lys Ile Pro ProPro Thr Arg Pro Leu Pro Thr 290 295 300 305 GTG CCC CCA CAC CGC TCT ATTCCT CCG GCT GAC CCA AGG AAA AAT GAC 1075 Val Pro Pro His Arg Ser Ile ProPro Ala Asp Pro Arg Lys Asn Asp 310 315 320 AGG CCA AAA CCT CCT CGG CCTCCA ACC GGC AGA CCC TCC TAT CCC GGA 1123 Arg Pro Lys Pro Pro Arg Pro ProThr Gly Arg Pro Ser Tyr Pro Gly 325 330 335 GCC AAA CCC AAC ATC TGT GATGGG AAC TTT AAC ACT CTA GCT ATT CTT 1171 Ala Lys Pro Asn Ile Cys Asp GlyAsn Phe Asn Thr Leu Ala Ile Leu 340 345 350 CGT CGT GAG ATG TTT GTT TTCAAG GAC CAG TGG TTT TGG CGA GTG AGA 1219 Arg Arg Glu Met Phe Val Phe LysAsp Gln Trp Phe Trp Arg Val Arg 355 360 365 AAC AAC AGG GTG ATG GAT GGATAC CCA ATG CAA ATT ACT TAC TTC TGG 1267 Asn Asn Arg Val Met Asp Gly TyrPro Met Gln Ile Thr Tyr Phe Trp 370 375 380 385 CGG GGC TTG CCT CCT AGTATC GAT GCA GTT TAT GAA AAT AGC GAC GGG 1315 Arg Gly Leu Pro Pro Ser IleAsp Ala Val Tyr Glu Asn Ser Asp Gly 390 395 400 AAT TTT GTG TTC TTT AAAGGT AAC AAA TAT TGG GTG TTC AAG GAT ACA 1363 Asn Phe Val Phe Phe Lys GlyAsn Lys Tyr Trp Val Phe Lys Asp Thr 405 410 415 ACT CTT CAA CCT GGT TACCCT CAT GAC TTG ATA ACC CTT GGA AGT GGA 1411 Thr Leu Gln Pro Gly Tyr ProHis Asp Leu Ile Thr Leu Gly Ser Gly 420 425 430 ATT CCC CCT CAT GGT ATTGAT TCA GCC ATT TGG TGG GAG GAC GTC GGG 1459 Ile Pro Pro His Gly Ile AspSer Ala Ile Trp Trp Glu Asp Val Gly 435 440 445 AAA ACC TAT TTC TTC AAGGGA GAC AGA TAT TGG AGA TAT AGT GAA GAA 1507 Lys Thr Tyr Phe Phe Lys GlyAsp Arg Tyr Trp Arg Tyr Ser Glu Glu 450 455 460 465 ATG AAA ACA ATG GACCCT GGC TAT CCC AAG CCA ATC ACA GTC TGG AAA 1555 Met Lys Thr Met Asp ProGly Tyr Pro Lys Pro Ile Thr Val Trp Lys 470 475 480 GGG ATC CCT GAA TCTCCT CAG GGA GCA TTT GTA CAC AAA GAA AAT GGC 1603 Gly Ile Pro Glu Ser ProGln Gly Ala Phe Val His Lys Glu Asn Gly 485 490 495 TTT ACG TAT TTC TACAAA GGA AAG GAG TAT TGG AAA TTC AAC AAC CAG 1651 Phe Thr Tyr Phe Tyr LysGly Lys Glu Tyr Trp Lys Phe Asn Asn Gln 500 505 510 ATA CTC AAG GTA GAACCT GGA CAT CCA AGA TCC ATC CTC AAG GAT TTT 1699 Ile Leu Lys Val Glu ProGly His Pro Arg Ser Ile Leu Lys Asp Phe 515 520 525 ATG GGC TGT GAT GGACCA ACA GAC AGA GTT AAA GAA GGA CAC AGC CCA 1747 Met Gly Cys Asp Gly ProThr Asp Arg Val Lys Glu Gly His Ser Pro 530 535 540 545 CCA GAT GAT GTAGAC ATT GTC ATC AAA CTG GAC AAC ACA GCC AGC ACT 1795 Pro Asp Asp Val AspIle Val Ile Lys Leu Asp Asn Thr Ala Ser Thr 550 555 560 GTG AAA GCC ATAGCT ATT GTC ATT CCC TGC ATC TTG GCC TTA TGC CTC 1843 Val Lys Ala Ile AlaIle Val Ile Pro Cys Ile Leu Ala Leu Cys Leu 565 570 575 CTT GTA TTG GTTTAC ACT GTG TTC CAG TTC AAG AGG AAA GGA ACA CCC 1891 Leu Val Leu Val TyrThr Val Phe Gln Phe Lys Arg Lys Gly Thr Pro 580 585 590 CGC CAC ATA CTGTAC TGT AAA CGC TCT ATG CAA GAG TGG GTG TGATGTAGG 1942 Arg His Ile LeuTyr Cys Lys Arg Ser Met Gln Glu Trp Val 595 600 605 GTTTTTTCTTCTTTCTTTCT TTTGCAGGAG TTTGTGGTAA CTTGAGATTC AAGACAAGAG 2002 CTGTTATGCTGTTTCCTAGC TAGGAGCAGG CTTGTGGCAG CCTGATTCGG GGCTGACCTT 2062 TCAAACCAGAGGGTTGCTGG TCCTGCACAT GAGTGGAAAT ACACTCATGG GGAA 2116 607 Amino acidSingle Linear Protein Human 2 Met Ile Leu Leu Thr Phe Ser Thr Gly ArgArg Leu Asp Phe Val His 1 5 10 15 His Ser Gly Val Phe Phe Leu Gln ThrLeu Leu Trp Ile Leu Cys Ala 20 25 30 Thr Val Cys Gly Thr Glu Gln Tyr PheAsn Val Glu Val Trp Leu Gln 35 40 45 Lys Tyr Gly Tyr Leu Pro Pro Thr AspPro Arg Met Ser Val Leu Arg 50 55 60 Ser Ala Glu Thr Met Gln Ser Ala LeuAla Ala Met Gln Gln Phe Tyr 65 70 75 80 Gly Ile Asn Met Thr Gly Lys ValAsp Arg Asn Thr Ile Asp Trp Met 85 90 95 Lys Lys Pro Arg Cys Gly Val ProAsp Gln Thr Arg Gly Ser Ser Lys 100 105 110 Phe His Ile Arg Arg Lys ArgTyr Ala Leu Thr Gly Gln Lys Trp Gln 115 120 125 His Lys His Ile Thr TyrSer Ile Lys Asn Val Thr Pro Lys Val Gly 130 135 140 Asp Pro Glu Thr ArgLys Ala Ile Arg Arg Ala Phe Asp Val Trp Gln 145 150 155 160 Asn Val ThrPro Leu Thr Phe Glu Glu Val Pro Tyr Ser Glu Leu Glu 165 170 175 Asn GlyLys Arg Asp Val Asp Ile Thr Ile Ile Phe Ala Ser Gly Phe 180 185 190 HisGly Asp Ser Ser Pro Phe Asp Gly Glu Gly Gly Phe Leu Ala His 195 200 205Ala Tyr Phe Pro Gly Pro Gly Ile Gly Gly Asp Thr His Phe Asp Ser 210 215220 Asp Glu Pro Trp Thr Leu Gly Asn Pro Asn His Asp Gly Asn Asp Leu 225230 235 240 Phe Leu Val Ala Val His Glu Leu Gly His Ala Leu Gly Leu GluHis 245 250 255 Ser Asn Asp Pro Thr Ala Ile Met Ala Pro Phe Tyr Gln TyrMet Glu 260 265 270 Thr Asp Asn Phe Lys Leu Pro Asn Asp Asp Leu Gln GlyIle Gln Lys 275 280 285 Ile Tyr Gly Pro Pro Asp Lys Ile Pro Pro Pro ThrArg Pro Leu Pro 290 295 300 Thr Val Pro Pro His Arg Ser Ile Pro Pro AlaAsp Pro Arg Lys Asn 305 310 315 320 Asp Arg Pro Lys Pro Pro Arg Pro ProThr Gly Arg Pro Ser Tyr Pro 325 330 335 Gly Ala Lys Pro Asn Ile Cys AspGly Asn Phe Asn Thr Leu Ala Ile 340 345 350 Leu Arg Arg Glu Met Phe ValPhe Lys Asp Gln Trp Phe Trp Arg Val 355 360 365 Arg Asn Asn Arg Val MetAsp Gly Tyr Pro Met Gln Ile Thr Tyr Phe 370 375 380 Trp Arg Gly Leu ProPro Ser Ile Asp Ala Val Tyr Glu Asn Ser Asp 375 390 395 400 Gly Asn PheVal Phe Phe Lys Gly Asn Lys Tyr Trp Val Phe Lys Asp 405 410 415 Thr ThrLeu Gln Pro Gly Tyr Pro His Asp Leu Ile Thr Leu Gly Ser 420 425 430 GlyIle Pro Pro His Gly Ile Asp Ser Ala Ile Trp Trp Glu Asp Val 435 440 445Gly Lys Thr Tyr Phe Phe Lys Gly Asp Arg Tyr Trp Arg Tyr Ser Glu 450 455460 Glu Met Lys Thr Met Asp Pro Gly Tyr Pro Lys Pro Ile Thr Val Trp 455470 475 480 Lys Gly Ile Pro Glu Ser Pro Gln Gly Ala Phe Val His Lys GluAsn 485 490 495 Gly Phe Thr Tyr Phe Tyr Lys Gly Lys Glu Tyr Trp Lys PheAsn Asn 500 505 510 Gln Ile Leu Lys Val Glu Pro Gly His Pro Arg Ser IleLeu Lys Asp 515 520 525 Phe Met Gly Cys Asp Gly Pro Thr Asp Arg Val LysGlu Gly His Ser 530 535 540 Pro Pro Asp Asp Val Asp Ile Val Ile Lys LeuAsp Asn Thr Ala Ser 545 550 555 560 Thr Val Lys Ala Ile Ala Ile Val IlePro Cys Ile Leu Ala Leu Cys 565 570 575 Leu Leu Val Leu Val Tyr Thr ValPhe Gln Phe Lys Arg Lys Gly Thr 580 585 590 Pro Arg His Ile Leu Tyr CysLys Arg Ser Met Gln Glu Trp Val 595 600 605 20 Nucleic acid SingleLinear Other nucleic acid unknown 3 SGNVVNGCWG AYATMRTSAT 20 27 Nucleicacid Single Linear Other nucleic acid unknown 4 YTCRTSNTCR TCRAARTGRRHRTCYCC 27 14 Amino acid Single Linear Peptide unknown 5 Gln Thr Arg GlySer Ser Lys Phe His Ile Arg Arg Lys Arg 1 5 10 14 Amino acid SingleLinear Peptide unknown 6 Glu Glu Val Pro Tyr Ser Glu Leu Glu Asn Gly LysArg Asp 1 5 10 18 Amino acid Single Linear Peptide unknown 7 Pro Thr SerPro Arg Met Ser Val Val Arg Ser Ala Glu Thr Met Gln 1 5 10 15 Ser Ala 14Amino acid Single Linear Peptide unknown 8 Thr Leu Gly Asn Pro Asn HisAsp Gly Asn Asp Leu Phe Leu 1 5 10 7 Amino acid Single Linear Peptideunknown 9 Gly Glu Ala Asp Ile Leu Val 1 5 9 Amino acid Single LinearPeptide unknown 10 Gly Asp Ala His Phe Asp Asp Asp Glu 1 5 7 Amino acidSingle Linear Peptide unknown 11 Gly Glu Ala Asp Ile Met Ile 1 5 7 Aminoacid Single Linear Peptide unknown 12 Pro Arg Cys Gly Val Pro Asp 1 5 24Amino Acid Single Linear Peptide unknown 13 Ala Ile Ala Ile Val Ile ProCys Ile Leu Ala Leu Cys Leu Leu 1 5 10 15 Val Leu Val Tyr Thr Val PheGln Phe 20 4 Amino Acid Single Linear Peptide unknown 14 Arg Xaa Lys Arg

What is claimed is:
 1. An isolated matrix metalloproteinase (MMP)protein or a salt thereof comprising the following peptide fragments ofSEQ ID No: 2: (a) Gly¹⁰⁹ to Arg¹¹⁹, (b) Pro¹⁷¹ to Gly¹⁷⁸, (c) Thr²²⁹ toLeu²⁴² and (d) Asp⁵³³ to Val⁶⁰⁷, said matrix metalloproteinase proteinhaving a maximum molecular weight of approximately 69 kDa and is a proMMP-2 activating factor.
 2. The protein or salt thereof according toclaim 1, further comprising the peptide fragment Ala⁵⁶⁴ to Phe⁵⁸⁷ of SEQID No: 2, said Ala⁵⁶⁴ to Phe⁵⁸⁷ fragment being located at or near theC-terminal of the protein.
 3. An isolated polynucleotide encoding theprotein according to claim 1 or
 2. 4. The polynucleotide according toclaim 3, which comprises a cDNA encoding the protein.
 5. Thepolynucleotide according to claim 4, comprising the nucleic acidsequence of SEQ ID No:
 1. 6. A vector comprising the polynucleotideaccording to claim
 4. 7. A host cell transformed or transfected with thevector according to claim
 6. 8. The polynucleotide according to claim 4,which is labeled.
 9. A probe for hybridization comprising thepolynucleotide according to claim
 8. 10. The polynucleotide according toclaim 3, comprising the nucleic acid sequence of SEQ ID No:
 1. 11. Avector comprising the polynucleotide according to claim
 3. 12. A hostcell transformed or transfected with the vector according to claim 11.13. The polynucleotide according to claim 3, which is labeled.
 14. Aprobe for hybridization comprising the polynucleotide according to claim13.
 15. The protein or salt thereof according to claim 1 or 2, which islabeled.
 16. The protein or salt thereof according to claim 1, havingthe amino acid sequence as set forth in SEQ ID No:
 2. 17. The protein orsalt thereof according to claim 1, which is obtained by prokaryotic oreukaryotic expression of an exogenous DNA sequence.
 18. The protein orsalt thereof according to claim 1, wherein the pro MMP-2 activatingfactor is one which is capable of converting pro-matrixmetalloproteinase 2 from its latent form to its active form.
 19. Aprocess for producing a matrix metalloproteinase protein or a saltthereof, comprising: culturing a host cell transformed or transfectedwith a vector comprising a polynucleotide which encodes the matrixmetalloproteinase protein or salt thereof of claim 30, in a nutrientmedium, and obtaining from the nutrient medium the matrixmetalloproteinase protein or salt thereof.